進階搜尋

 
查詢範圍:「   」
顯示範圍:第筆 論文書目資料
顯示格式:全部欄位
共 13 筆
------------------------------------------------------------------------ 第 1 筆 ---------------------------------------------------------------------
系統識別號 U0007-0402200913180200
論文名稱(中文) 應用減少腦部神經膠質細胞纖維化提升腦部氧氣分壓與維持最佳腦部血液灌流壓於治療嚴重頭部外傷之研究
論文名稱(英文) Application of Decreasing Brain Tissue Gliosis, Elevating Brain Tissue O2 Partial Pressure, and Maintaining Optimal Cerebral Perfusion Pressure in Treatment of Severe Traumatic Brain Injury
校院名稱 臺北醫學大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Graduate Institute of Clinical Medicine
學年度 97
學期 1
出版年 98
研究生(中文) 林家瑋
學號 D002089012
學位類別 博士
語文別 中文
口試日期 2008-12-09
論文頁數 126頁
口試委員 指導教授-邱文達教授
委員-鄭瑞堂教授
委員-林茂村教授
委員-邱弘毅教授
委員-蔡行瀚教授
關鍵字(中) 嚴重頭部外傷
神經膠質細胞纖維化
腦部氧氣分壓
腦部血液灌流壓
關鍵字(英) Severe traumatic brain injury
gliosis
hyperboric oxygen therapy
Cerebral Perfusion Pressure
學科別分類
中文摘要 1997年以前,台灣地區的頭部外傷發生率、死亡率及嚴重度均為世界上最嚴重的地區,主要原因為機車的高使用率與安全帽的低配戴率。為了因應這個嚴重的問題,我們進行了一連串的研究。在過去二十年來把我們所進行的研究方法、對象及內容大致分成八大研究主題,分別簡述如下:(一)頭部外傷流行病學研究:一開始進行全國性頭部外傷病人收集,發現台灣地區頭部外傷的發生率為鄉村地區較高,且其中機車車禍導致頭部外傷佔了七成,研究中顯示機車騎士配戴安全帽的比例甚低,因此建議推動安全帽的強制立法是減輕及防止頭部外傷的最重要政策。(二)介入研究:本研究針對安全帽立法前後做一完整趨勢研究比較,並從中瞭解到安全帽的使用確實有其必要性。研究顯示機車事故中,在安全帽立法後,戴安全帽者發生意識喪失、顱骨骨折、顱內出血等情形,皆較立法前少。顯示安全帽的立法確有其功效。(三)神經行為調查研究:本研究的目的,主要是將Levin et al.(1998)所發展的神經行為量表(Neurobehavioral Rating Scale,簡稱NRS),進行台灣地區的初步驗證工作,以瞭解頭部外傷病人之行為改變模式及與受傷嚴重性和持續性之間的關係。研究結果在不同程度的頭部外傷受試中,NRS的「認知語言」因素及「焦慮」因素上是有顯著差異的。(四)脊髓損傷流行病學研究:安全帽立法後,為探討創傷性頸脊髓損傷與機車安全帽使用的相關性。研究結論是安全帽並不會造成更高之頸脊髓損傷機率。(五)國際合作性研究:對於發展中國家而言,頭部傷害是一個複雜和困難的問題。這些國家通常都缺乏人力資源和設備。跟歐洲和美洲相較下,發展中國家還是有較大的頭部傷害問題。 (六)嚴重頭部外傷研究:腦部外傷是造成死亡與嚴重殘障的主要原因。根據美國的統計資料, 每一年的頭部外傷人數約為一百五十萬人,其中大約有一百萬人因為頭部外傷而到醫院的急診室就診及處理。其中總共住院的病人數約有二十三萬五千人,九萬名病人造成嚴重殘障。。另外每年因為腦部外傷而死亡的的人數大約有 5萬人。頭部外傷照護之主要目的是降低其死亡率及殘障程度,並盡可能的提高傷者之生活品質。對於嚴重之頭部外傷病患採用加護重症照顧可顯著降低死亡率及殘障程度,美國更於1995年正式發表嚴重頭部外傷處理準則(Guidelines of management in severe head injury),其中主張嚴重病患應裝置顱內壓(Intracranial pressure, ICP)監測,及腦灌流壓(Cerebral perfusion pressure, CPP)的維持,藉此以避免腦部缺血所造的細胞壞死,經過五年之評估,認為此一新觀念確實有其臨床效果。(七)輕度頭部外傷研究:本國際合作專案為一個跨國及跨文化之世代研究計畫,邀集澳洲、義大利、印度、美國、阿根廷及台灣六國共同參加。計畫於各國都以同樣的研究方法及標準化的表格進行同步研究。希望能藉此了解在不同的國家及不同的文化背景之下,輕度頭部外傷病患之傷害的早期指標與後期結果間的關係。 (八)嚴重頭部外傷診療指引研究:本研究邀請台灣地區神經科學領域北、中、南、東,台灣地區醫學中心及區域醫院共32位專家學者,經過多次的會議討論及研討會,共同制訂台灣版嚴重頭部外傷治療共識 (Guidelines)。依計畫之執行方向,製訂八大主題:1.急診處置 Management in ER (ER: Emergency Room). 2. 顱內壓監測 ICP monitor. 3. 腦灌流壓與輸液之原則 CPP and Fluid management. 4. 鎮定劑之使用 Sedation. 5. 營養 Nutrition. 6. 顱內壓上升之處置 Management of Intracranial Hypertension. 7. 癲癇之預防治療 Seizure Prophylaxis. 8. 二線療法 Second Tier Therapy
其中之嚴重頭部外傷研究部份正是本論文之研究主題,本論文之研究方向主要是真對四大方向進行相關之基礎及臨床研究,其結論與摘要如下:(甲)減少腦傷後癲癇發生之機率。本研究藉由玻尿酸與細胞表面上相關受器的交互作用,影響細胞的活動和增生能力。腦部損傷後的病人,恢復神經功能,多在受傷後半年內快速進行,然而在半年之後,隨著疤痕組織形成及穩定,神經功能的恢復也日趨穩定,也就是恢復的速度也日趨緩慢。在這次實驗中,實驗組中,無論是4週,8週,或是12週的老鼠,中樞神經系統受傷表面疤痕組織中GFAP+細胞的數目都較對照組的老鼠受傷腦部表面少。受傷後神經疤痕組織的厚度及神經膠質化的程度也較趨於緩和。這些增厚的疤痕組織常常被認為與阻礙神經發展恢復,甚至於被認為與頭部外傷後的癲癇,特別是慢性長期性的癲癇有關。 (乙)提升血液中之氧分壓。在本次試驗中,我們顯示出腦部外傷病人的GCS昏迷指數在接受高壓氧治療的族群比起控制組有顯著的進步。這個結果顯示出高壓氧對於腦部外傷病人GCS昏迷指數的恢復有正面的影響。關於GOS (Glasgow 預後量表)的進步上,接受高壓氧治療的病人,在三個月的追蹤兩組並沒有差別,但在高壓氧治療六個月後的追蹤,GOSb=4 的族群有得到一些進步。這表示高壓氧治療須要更多時間來表達影響,高壓氧可以幫助輕度神經功能缺失的病人恢復並回到日常生活,在這前瞻性的試驗中,我們可以定出以下結論,高壓氧治療可以改善腦部外傷病人的昏迷指數恢復同時幫助輕度神經功能殘障的病人恢復,而能享有較好的人生。(丙)盡快降低水腫之程度。血液透析不平衡症候群(dialysis disequilibrium syndrome-DDS)是一種在血液透析治療過程中,發生急性神經學功能上的異常表現。我們發現幾乎所有急性及慢性腎衰竭的病患,在接受血液透析的時候,都發現顱內壓上升的情形。在我們這個研究中,顱內壓的波動在血液透析治療過程中的第二個小時內急遽的上升,而且在滿第二個小時顱內壓上升到最高峰。在腦部手術後病患需要血液透析治療時。於本研究中藉由改變血液透析的治療方式,例如:減少每次血液透析的總量和流速,增加血液透析的頻率,達到更緩和的方式去清除尿素分子,降低腦內出血腎衰竭的病人產生血液透析不平衡症候群的機會。(丁)提升腦部血液之灌流量以減少腦傷後之二度傷害。我們的研究結果顯示,有放置顱內壓監測器的病患不但存活率較高,Glasgow outcome scale 也較佳,昏迷指數較差的病患,死亡率相對的較高,約在百分之八十左右。在昏迷指數三分到五分的這一組當中,維持腦灌流壓高於70毫米汞柱不但可以降低死亡率,對於短期及長期的預後也有改善。針對昏迷指數六分到八分者,將腦灌流壓維持在60毫米汞柱以上即足夠。針對嚴重腦幹功能損傷的病患,維持腦灌流壓高於70毫米汞柱可以明顯的改善預後。對於死亡率的控制更佳,若能夠避免因為要維持較高的腦灌流壓而發生的併發症,將腦部的灌流壓維持在70毫米汞柱以上對於腦外傷的病患來說應是有所助益的。
臨床上治療嚴重腦外傷的方法主要為:減少腦傷後癲癇發生之機率,提升血液中之氧分壓,盡快降低水腫之程度,及提升腦部血液之灌流量以減少腦傷後之二度傷害。以本論文之研究結論主要是真對上述之四大方向進行進一步之探討。
根據本論文之四大主題主要是針對嚴重頭部外傷診療指引建立相關之研究及理論基礎,希望借由本論文中粗淺且簡單之結論拋磚引玉,誘導國內從事相關研究工作之專家群能一起全心投入頭部外傷相關之研究,進而建立本土化之研究結論,如此才能完備建立嚴重頭部外傷診療指引時所需之所有基礎,也只有在具有本土化之研究結論的嚴重頭部外傷診療指引才有足夠之說服力去大力推行此診療指引在台灣地區甚或亞洲地區確實的落實。
英文摘要 Before 1997, Taiwan was one of the areas with the highest incidence and mortality rate of traumatic brain injuries (TBI) in the world. This situation mainly resulted from a large number of motorcyclists, of whom only very few wore a helmet. In order to tackle this serious problem, we carried out several studies on TBI in the past few years. According to different subjects, material and methods, there were seven main topics of studies, which were:1. Epidemiological study of TBI: We initiated a nationwide brain injury survey. We found that incidence of TBI was higher in the rural area than in the urban area of Taiwan. Seventy percent of the TBI were due to motorcycle accidents and most of the motorcycle riders did not wear a helmet. So we asserted that the mandatory helmet use law would be the most important and effective policy to reduce and to prevent TBI in Taiwan. 2. Intervention study of TBI: We started a complete study comparing the trend of TBI before and that after the mandatory helmet use law. We found that after the helmet use law, there was a reduction of the rate of consciousness disturbance, skull fracture and intracranial hemorrhage in the motorcycle related accidents. We concluded that the helmet use law intervention was effective and the continue education for the use of helmet and re-enforcement of the helmet use law were essential to maintain a satisfactory rate of helmet use in motorcyclists.
3. Study of neurobehavioral: With the use of the neurobehavioral rating scale (NRS) of Levin et al. (1987), we investigated the relationship of neurobehavioral disturbances in TBI patients with the severity and duration of head injury. NRS factors “cognitive language” and “anxiety” were significantly different in head injury subgroups with different degree of brain injury. 4. Epidemiological study of spinal cord injury: After the helmet use law, the incidence of TBI in Taiwan was proved to be successfully declined, but the use of helmet might increase the risk of spinal cord injury. In order to investigate the correlation of helmet use with spinal cord injury, we collected the registered and telephone interview data from the “Head and Spinal Cord Injury Study Group, R.O.C” for important after injury evaluation. The result showed that the use of helmet would not cause cervical spine injury. 5. International collaborative program: TBI is a tough and complicated issue for developing countries. However, most of these countries have limited resources and facilities for TBI prevention and treatment. Kraus, Jennett and Frankowski reported in 1990 that the annual incidence rate of head injury was 132–430/105, and the annual morality rate was 9–32/105. Although every country has their own method, definition and strategies in their TBI databases collection, the comparative results between developing countries and developed countries showed that the developing countries faced a more serious problem of TBI. 6. Quality of life study of severe TBI: The main goal of the TBI patients care is to reduce the severity of disability, mortality rates, and their quality of life as much as possible. Since 1970, many researches showed that application of intensive care on severe TBI patients significantly decreased the severity and mortality rates. USA published the “Guidelines of management in severe head injury” in 1995, suggesting the use of intracranial pressure monitors to control not only the intracranial pressure, but also to maintain the cerebral perfusion pressure. That was a breakthrough concept that could clinically help to avoid cell necrosis due to ischemia of brain tissues and was now widely used in Europe and America after a 5 years evaluation. 7. Study of minor TBI: This is an international collaborative cohort study of six countries (Australia, Italy, India, USA, Argentina and Taiwan) with standardized method of study and forms. We aim to find out the risk and prognostic index of patients with minor traumatic brain injury, and provide patients with better quality of care. 8. Guidelines for management of severe TBI in Taiwan: We invited 32 neurosurgical experts from medical centers and local regional hospitals in different parts of Taiwan to join the Taiwan TBI guideline consensus meetings and conferences. We chose eight topics of guideline development:
• Management in emergency room
• ICP monitor
• CPP and fluid management
• Use of sedation
• Nutrition
• Management of intracranial hypertension
• Seizure prophylaxis
Second tier therapy
Within this dissertation, 4 major basic and clinical researches for the severe traumatic brain injury were conducted. 4 major conclusions were summarized as following: (1) HA application could effectively reduce the gliosis in brain cortex not only on the thickness of the scarring and also in glia cell proliferation. All these change was believed to be related with the lesser chance of post traumatic or post operative seizure incidence. (2) HBOT could elevate the brain oxygenation; promote the GCS improvement, and also the 3 months outcome, esp. in GOS change. But the timing when the HOBT implement need further clarification. (3) Slower speed of hemodialysis, more frequently dialysis could reduce the severity of brain edema effective. These subtle changes for the patient who need dialysis could reduce IICP incidence and increase the survival rate for these patient also. And (4) Improved cerebral perfusion, esp. for the patient with extreme poor GCS after the brain trauma could be indicated for the better outcome and better survival rate. But the high complication rate should be also monitored closely to avoid the further disaster during the maintenance patient’s CPP.
These entire 4 major topics was only the beginning of the clinical trial for the development of the treatment guideline of severe traumatic brain injury. We hope in the near future, some more researchers could join into this field of research. With all there efforts evidence-based guideline could be then setup and well accepted by the clinical practice.
論文目次 縮寫表(Abbreviations) ……………………………………………001
中文摘要(Chinese abstract) ………………………………………004
英文摘要(English abstract) ………………………………………008
前言(Introduction): ………………………………………………012
(一)頭部外傷流行病學研究 …………………………………………012
(二)介入研究 …………………………………………………………013
(三)神經行為調查研究 ………………………………………………017
(四)脊髓損傷流行病學研究 …………………………………………018
(五)國際合作性研究 …………………………………………………020
(六)嚴重頭部外傷病患生活品質之研究 ……………………………020
一、神經外科醫師訪視 ……………………………………………022
二、全國頭部外傷資料登錄 ………………………………………023
三、嚴重頭部外傷病患ICU照護追蹤 ……………………………023
四、嚴重頭部外傷病患生活品質之追蹤 …………………………023
(七) 輕度頭部外傷研究 ……………………………………………026
(八)嚴重頭部外傷診療指引研究 ……………………………………027
(九)頭部外傷之基礎與臨床研究 ……………………………………029
(甲)減少腦傷後癲癇發生之機率 .…………………………………030
(乙)提升血液中之氧分壓並降低水腫之程度 .……………………033
(丙)盡快降低水腫之程度 ………………………………..…………034
(丁)提升腦部血液之灌流量以減少腦傷後之二度傷害 …….……035
減少腦傷後癲癇發生之基礎研究 ……………………………………036
研究方法與材料 ……………………………………………………036
實驗設計和手術方法 ………..…….………………………………036
組織學的檢查 ………….…………………………………………037
資料的分析 …………….…………………………………………038
結果 …………………………………………………………………038
提升血液中之氧分壓並降低水腫之臨床試驗 ……………………040
緒論 …………………….……………………………………………040
研究疾病的目前現況……………………………………….………040
自然病程…………………………………….………………………040
研究方法與材料 ………………………………………………………044
結果…………………………………………………………………046
不良反應…………………………………………………………047
降低外傷後腦部組織水腫之臨床試驗 ……………………………049
研究方法與材料 ……………………………………………………049
病患的臨床表現 ……………………………………………………049
血液透析計畫 ……...………………………………………….……051
結果 ……………………………………………………………………051
提升腦部血液之灌流量以減少二度傷害之臨床試驗 ………………053
緒論 ……….………………………….……………………………053
研究方法與材料 …………………………..……………………054
結果 …………………………………….……………………………056
討論與結論…………………………...……………………………059
研究限制 ……………………………………………….……………066
總結 ……………………………………………………………………068
感謝 …………………………………………………………………070
參考文獻 ……………………………….…………………………071
圖表 ………….……………………………………………………080
附件 ……………………………………………………………………092
參考文獻 1.Alvarez M, Nava JM, Rue M, Quintana S. (1998) Mortality prediction in head trauma patients “ performance of Glasgow coma scale and general severity systems. Critical care Med. 26(1): 142-148.
2.Arieff A, Massry S, Barrientos A, Kleeman C 1973: Brain water and electrolyte metabolism in uremia: Effects of slow and rapid hemodialysis. Kidney Internat, 4:177-187.
3.Arieff A, Guisade R, Massry S, Lazarowitz V 1976: Central nervous system pH in uremia and the effects of hemodialysis. J Clin Invest, 58:306-311.
4.Arieff A: Dialysis disequilibrium syndrome: Current concepts on pathogenesis and prevention. Kidney Internat 1994, 45:629-635.
5.Artru F, Chacornac R., Deleuze R. (1976) Hyperbaric oxygenation for severe head injuries: preliminary results of a controlled study. Eur Neurological Research. 14: 310-318.
6.Bader M, Littlejohns L, March K. (2003) Brain tissue oxygen monitor in severe brain injury, II Implication for critical care team. Critical care nurse. 23: 29-43.
7.Bennett MH, Trytko B, Jonker B 2004 Oct. Hyperbaric oxygen therapy for the adjunctive treatment of traumatic brain injury. Cochrane Database Syst Rev. 18:CD004609.
8.Bignami A, Hosley M, Dahl D 1993. Hyaluronic acid and hyaluronic acid-binding proteins in brain extracellular matrix. Anat Embryol (Berl);188:419-33.
9.Bulger EM, Natherns AB, Rivara FP et al. (2002) The Brain Trauma Foundation. Management of Severe Head Injury:institutional variation in care and effect on outcome. Critical care medicine. 30: 1870-1876.
10.Bullock R, Chesnut RM, Clifton G, et al 1996. Guidelines for the management of severe head injury. Brain Trauma Foundation. Eur J Emerg Med.;3:109 –127.
11.Chia-Ying Kuo, Wen-Ta Chiu, Kau-Hsun Yeh, et al. 1996: Spinal Cord Injury in the Elderly. Journal of Taipei Medical College, 25: 22-26.
12.Chia-Ying Kuo, Wen-Ta Chiu, Chi-Chun Hung, et al. 1998: The effect of the Taiwan motorcycle helmet law on motorcycle crash fatalities and head injuries. Journal of Taipei Medical College, 27: 1-8.
13.Ching-Chang Hung, Wen-Ta Chiu, Jui-Chang Tsai, Ronald E. LaPorte and Chun-Jen Shih 1991: An Epidemiological Study of Head Injury in Hualien County, Taiwan. Journal of Formosan Medical Association, 90: 1227-33.
14.Ching-Chang Hung, Wen-Ta Chiu, and Head & Spinal Cord Injury Research Group: Developing An Early Management Program For Acute Head Injury. Proceedings of International Conference on Recent Advances in Neurotraumatology, Gold Coast, Queensland, Australia , 41-42. 1994.
15.Contant CF, Valadka AB, Gopinath SP, et al. (2001) Adult respiratory distress syndrome: A complication of the induced hypertension after head injury. Journal of Neurosurgery. 95(4): 560-568.
16.Czosnyka M, Balestreri M, Steiner L, et al. Age, intracranial pressure, autoregulation, and outcome after brain trauma. J Neurosurg 2005; 102: 450–4
17.Dan-Min Wu, Wen-Ta Chiu, Ching-Chung Hung: A Study of Head Injury Among Teenagers. Journal of Medical Sciences, 12(2): 104-115, 1991.
18.DiFresco V, Landman M, Jaber B, White A: Dialysis disequilibrium syndrome: an unusual cause of respiratory failure in the medical intensive care unit. Intensive Care Medicine 2000, 26:628-630.
19.Eben Alexander (Wen-Ta Chiu and Ronald E. LaPorte): Editorial: A Briefing of International Symposium of Epidemiology of Head and Spinal Cord Injury and Global SHIP Program. Surgical Neurology, 38:478-9, 1992.
20.Feigin I. The mucopolysaccharides of the ground substance of the human brain. J Neuropathol Exp Neurol 1980;39(1):1-12.
21.Fiesseler FW, Silverman ME, Riggs RL, Szucs PA. (2006) Indication for hyperbaric oxygen treatment as a predictor of tympanostomy tube placement. Undersea Hyperb Med. 33(4): 231-235.
22.Fox K, Caterson B. Neuroscience. Freeing the bain from the perineuronal net. Science 2002;298(5596):1187-9.
23.Frey LC. Epidemiology of posttraumatic epilepsy: a critical review. Epilepsia 2003;44 Suppl 10:11-7.
24.Girard N, Delpech A, Delpech B. Characterization of hyaluronic acid on tissue sections with hyaluronectin. J Histochem Cytochem 1986;34(4):539-41.
25.Golden ZL, Neubauer R, Golden CJ, Greene L, Marsh J, Mleko A. (2002) Improvement in cerebral metabolism in chronic brain injury after hyperbaric oxygen therapy. Int J Neurosci. 112(2): 119-131.
26.Hardy P, Johnston KM, De Beaumont L, Montgomery DL, Lecomte JM, Soucy JP, Bourbonnais D, Lassonde M. (2007) Pilot case study of the therapeutic potential of hyperbaric oxygen therapy on chronic brain injury. J Neurol Sci. 253(1-2): 94-105.
27.Harris C, Townsend J: Dialysis disequilibrium syndrome: Clinicopathologic conference. West J Med 1989, 151:52-55.
28.Haw-Ming Huang, Maw-Chang Lee, Wen-Ta Chiu, Chien-Tsu Chen, and Sheng-Yang Lee: Three-Dimensional Finite Element Analysis of Subdural Hematoma. The Journal of Trauma: Injury, Infection, and Critical Care, 47(3): 538-544, 1999.
29.Horn CE, Himel HN, Selesnick SH. Hyperbaric oxygen therapy for sudden sensorineural hearing loss: a prospective trial of patients failing steroid and antiviral treatment. Otol Neurotol. 2005 Sep;26(5):882-9.
30.Hui-Wen Lin, Wen-Ta Chiu, Chia-Ying Kuo, Wen-Yin Hsueh: A Descriptive Epidemiological Study of Head Injury among Adolescents. Journal of Taipei Medical College, 24(12): 29-35, 1995.
31.Hsin-Ying Chen, Wen-Ta Chiu, Shun-Sheng Chen, Liang-Shong Lee, Chun-I Hung, Chun-Long Hung, Yeou-Chih Wang, Ching-Chang Hung, Lieh-Sheng Lin, Yang-Hsin Shih: A nationwide epidemiological study of spinal cord injuries in Taiwan from July 1992 to June 1996. Neurological Research, 19:617-622, 1997.
32.Hsin-Ying Chen, Shun-Sheng Chen, Wen-Ta Chiu, Liang-Shong Lee, Chun-I Hung, Chun-Long Hung, Yeou-Chih Wang, Ching-Chang Hung, Lieh-Sheng Lin, Yang-Hsin Shih, Chia-Ying Kuo: A Nationwide Epidemiological Study of Spinal Cord Injuries in Geriatric Patients in Taiwan. Neuroepidemiology, 16:241-247, 1997.
33.Hu M, Bankir L, Michelet S, Rousselet G, Trinh-Trang-Tan M: Massive reduction of urea transporters in remnant kidney and brain in uremic rats. Kidney Internat 2000, 58:1202-1210.
34.Jia-Ying Guo, Wen-Ta Chiu, Wen-Yin Hsueh, Hui-Wen Lin: A Study of Head injury and Motorcycle Accident in Taiwan. Journal of Taipei Medical College, 24(12): 36-43, 1995.
35.Kernie SG, Erwin TM, Parada LF. Brain remodeling due to neuronal and astrocytic proliferation after controlled cortical injury in mice. J Neurosci Res 2001;66(3):317-26.
36.Kishimoto T, Yamagami S, Tanaka H, Ohyama T, Yamamoto T, Yamakawa M, Nishino M, Yoshimoto S, Maekawa M: Superiority of hemofiltration to hemodialysis for treatment of chronic renal failure: comparative studies between hemofiltration and hemodialysis on dialysis disequilibrium syndrome. Artif Organs 1980, 4:86-93.
37.Langlois JA, Kegler SR, Butler JA. (2004) Traumatic brain injury in United States: Emergency department visits, hospitalization and deaths.
38.Lannoo E, Colardyn F, De deyne, et al. (1998) Cerebral perfusion pressure and intracranial pressure in relation to neuropsychological outcome. Intensive care Med. 24(3): 236-241.
39.Levin HS. Cognitive function outcomes after traumatic brain injury. Curr Opin Neurol 1998;11(6):643-6.
40.Liang-Shong Lee, Yang-Hsin Shih, Wen-Ta Chiu, Lieh-Sheng Lin, Chun-Ming Wu, Yeou-Chih Wang, Jan-Shan Huang, Ching-Chang Hung, Chun-Jen Shih: Epidemiologic Study of Head Injuries in Taipei City, Taiwan. Chinese Medical Journal (Taipei), 50:219-25, 1992.
41.Management and Prognosis of Severe Traumatic Brain Injury: guideline for the management of severe head injury. Brain trauma Foundation, 2000.
42.Marshall M, Golper T, Shaver M, Alam M, Chatoth D: Sustain lowefficiency dialysis for critically ill patients requiring renal replacement therapy. Kidney Intern 2001, 60:777-785.
43.Marshall LF. Smith RW. Shapiro HM. (1979) The outcome with aggressive treatment in severe head injury part I:the significant of intracranial pressure monitor. Journal of Neurosurgery. 50(1):20-25.
44.Marshall M, Golper T, Shaver M, Alam M, Chatoth D: Urea kinetics during sustained low-efficiency dialysis in critically ill patients requiring renal replacement therapy. Am J Kidney Dis 2002, 39:556-570.
45.McDonagh M, Helfand M, Carson S, Russman BS. Hyperbaric oxygen therapy for traumatic brain injury: a systematic review of the evidence. Arch Phys Med Rehabil. 2004 Jul;85(7):1198-204.
46.Meirovithz E, Soon J., Mayevsky A (2007) Effect of hyperbaric oxygenation on brain hemodynamics, hemoglobin oxygenation and mitochondrial NADH. Brain research Reviews. S4: 294-304.
47.Meyer K PJ. Polysaccharide of vitreous humor. J Biol Chem 1934.;107:629-34.
48.Mau-Roung Lin, Hei-Fen Hwang, Chih-Yi Chen, Wen-Ta Chiu(2007) Comparisons of the brief form of the World Health Organization Quality of Life and Short Form-36 for persons with spinal cord injuries. [Comparative Study. Journal Article. American Journal of Physical Medicine & Rehabilitation 86(2): 104-113.
49.Ming-Dar Tsai, Wen-Ta Chiu, Jia-Wei Lin, Chun-Fu Chen, Sheng-Jean Huang, Cheng-Kuei Chang, Wan-Lin Chen, Shin-Han Tsai (2006) Current experiences in the use of the severe head-injury guidelines in Taiwan. Surgical Neurology 66 (Suppl 2): S3-7.
50.NIH, Traumatic Brain Injury: Hope Through Research. 2002.
51.Ogawa M. Rating severity of the injury by ambulance aftendants J. Trauma. 1979;14:934.
52.Pei-Yeh Chiang, Wan-Chen Tsai, Wen-Ta Chiu, Chun-Huang Huang, Cheuk-Sing Choy, Wai-Mau Choi, Shin Han Tsai, Hung-Yi Chiou: National Traumatic Brain Injury in Preschool Children in Tiawan. New Taipei Journal of Medicine. 4 (4): 235-242, 2002.
53.Parent JM, Yu TW, Leibowitz RT, Geschwind DH, Sloviter RS, Lowenstein DH. Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus. J Neurosci 1997;17(10):3727-38.
54.Perides G, Biviano F, Bignami A. Interaction of a brain extracellular matrix protein with hyaluronic acid. Biochim Biophys Acta 1991;1075(3):248-58.
55.Peterson H: Acute encephalopathy occurring during hemodialysis. Arch Intern Med 1964, 113:877-880.
56.Robertson CS, Valadka AB, Hannay HJ, et al 1999. Prevention of secondary ischemic insults after severe head injury. Crit Care Med; 27: 2086–95
57.Rockswold GL, Ford SE, Anderson DC, Bergman TA, Sherman RE. (1992) Results of a prospective randomized trial for treatment of severely brain-injured patients with hyperbaric oxygen. J Neurosurg. 76: 929-934.
58.Rockswold SB, Rockswold GL, Defillo A. (2007) Hyperbaric oxygen in traumatic brain injury. Neurological Research. 2: 162-172.
59.Rosner MJ. Rosner SD. Johnson AH. (1995) Cerebral perfusion pressure:management protocol and clinical results. Journal of Neurosurgery. 83: 949-962.
60.Ruoslahti E1996. Brain extracellular matrix. Glycobiology;6:489-92.
61.Sbordone RJ, Liter JC, Pettler-Jennings P1995. Recovery of function following severe traumatic brain injury: a retrospective 10-year follow-up. Brain Inj;9:285-99.
62.Shin-Yuan Chen, Chun-Yuan Cheng, Chain-Fa Su, Yet-Wen Chen, Yu-Jen Yang, Yuh-Lin Chang, Wen-Ta Chiu 2000: Spectrum of Spontaneous Intracerebral Hemorrhage in Eastern Taiwan. Acta Neurologica Taiwanica, 9: 235-242.
63.SHIP's Crew (Coordinators: Wen-Ta Chiu, Ronald E. LaPorte) 1993: Global Spine and Head Injury Prevention (SHIP) Project. Proceeding of First International Symposium of Epidemiology of Head & Spinal Cord Injury, 97-104.
64.Schootman M, Buchman T. Lewis L (2003) National estimates of hospitalization charges for acute care of traumatic brain injuries. Brain injury. 17: 983-990.
65.Silver S: Cerebral edema after rapid dialysis is not caused by an increase in brain organic osmolytes. J Am Soc Nephrol 1995, 6:1600-1606.
66.Silver S, DeSimone J Jr, Smith D, Sterns R: Dialysis disequilibrium syndrome (DDS) in the rat: Role of the "reverse urea effect". Kidney Internat 1992, 42:161-166.
67.Silver S, Sterns R, Halperin M: Brain swelling after dialysis: Old urea or new osmoles? Am J Kidney Dis 1996, 28:1-13.
68.Sosin D. Sniezek J. Waxweiler R. (1995) Trends in death associated with traumatic brain injury 1979 through 1992. Success and failure. JAMA . 273(22): 1778-1780.
69.Su-Meei Lin, Wen-Ta Chiu, Ya-Li Hung, Ching-Lin Hong, etc: A Descriptive Epidemiological Study of Spinal Cord Injury. Journal of Taipei Medical College, 25(1): 27-32, 1996.
70.The BTF & HANS: Abstracts of the 5th International Neurotrauma Symposium, the point section on Neurotrauma and critical care. Guideline for cerebral perfusion pressure. Restorative Neurology and Neuroscience .16(3~4):149~319.
71.Thurman D, Alverson C, Guerrero J et al (1999) Traumatic Brain injury in the United States:A public health perspective. Journal of head trauma Rehabilitation. 14(6): 602-615.
72.Trachtman H, Futterweit S, Tonidandel W, Gullans S: The role of organic osmolytes in the cerebral cell volume regulatory response to acute and chronic renal failure. J Am Soc Nephrol 1993, 3:1913-1919.
73.Trochon V, Mabilat C, Bertrand P, Legrand Y, Smadja-Joffe F, Soria C, Delpech B, Lu H. Evidence of involvement of CD44 in endothelial cell proliferation, migration and angiogenesis in vitro. Int J Cancer 1996;66(5):664-8.
74.Updated CPP Guidelines approved By the AANS on March14, 2003.
75.Wen-Ta Chiu: The Epidemiology of Head Injury in Hualien County, Taiwan. Doctoral Dissertation of University of Pittsburgh, Jun 1989.
76.Wen-Ta Chiu, Ching-Chang Hung, Lieh-Hsien Lin, et al.: Epidemiology of Head Injury in Taiwan Area. Journal of Taipei Medical College, 19:107-111, 1990.
77.Wen-Ta Chiu: Epidemiology of Head Injury - A Review of International Studies. Journal of Taipei Medical College, 21:1-16, 1992.
78.Wen-Ta Chiu, Ching-Chung Hung, Lieh-Sheng Lin, Chun-Jen Shih, Ronald E. LaPorte: Hospitalized Head Injuries in Taipei City, Taiwan, 1983. Journal of Taipei Medical College, 21:17-23, 1992.
79.Wen-Ta Chiu: A Briefing of the International Symposium of Epidemiology of Head and Spinal Cord Injury in Developing Country. Journal of Taipei Medical College, 21: 105-107, 1992.
80.Wen-Ta Chiu, Stephen Deareater, Daniel McCarty, Thomas Songer, Ronald E. LaPorte: Establishment of Accurate Incidence Rates for Head and Spinal Cord Injuries in Developing and Developed Countries: A Capture-Recapture Approach. Journal of Trauma, 35: 206-11, 1993.
81.Wen-Ta Chiu, Wen-Ying Lin, Lieh-Sheng Lin, Ching-Chang Hung, Chun-Jen Shih: Neurobehavioral Manifestations Following Closed Head Injury. Journal of Formosan Medical Association, 92(3): 255-62, 1993.
82.Wen-Ta Chiu, Ching-Chang Hung, Liang-Shong Lee, Lieh-Sheng Lin, Chun-Jen Shih, Ronald E. LaPorte: Comparative Study of Head Injury in Urban & Rural Population in Taiwan. Proceeding of First International Symposium of Epidemiology of Head & Spinal Cord Injury, 129-136, 1993.
83.Wen-Ta Chiu, Ching-Chang Hung, Liang-Shong Lee, Lieh-Sheng Lin, Chun-Jen Shih, Ronald E. LaPorte. An Epidemiological Study of Spinal Cord Injury. Proceeding of First International Symposium of Epidemiology of Head & Spinal Cord Injury, 139-142, 1993.
84.Wen-Ta Chiu, Ching-Chang Hung, Liang-Shong Lee, Lieh-Sheng Lin, Chun-Jen Shih, Ronald E. LaPorte. Registry of Head Injury in Eastern Part of Taiwan & Penghu Hsien. Proceeding of First International Symposium of Epidemiology of Head & Spinal Cord Injury, 137-138, 1993.
85.Wen-Ta Chiu, Ronald E. LaPorte: SHIP Notice. Surgical Neurology, 39: 415, 1993.
86.Wen-Ta Chiu, Ronald E. LaPorte: Global Spine and Head Injury Prevention (SHIP) Project (Letter to the editor). Journal of Trauma, 35: 969-970, 1993.
87.Wen-Ta Chiu, Ronald E. LaPorte, G. Gururaj, Iftikhar Ali Raja, Thomas J. Pentel'enyi, Bouyoucef, Arnoldo Levy: Head Injury In Developing Countries. Neurotrauma , Sept 1993.
88.Wen-Ta Chiu, Jia-Wei Lin, Ching-Chang Hung, and Head & Spinal Cord Injury Research Group: Head Injury in Urban and Rural Populations in Taiwan. Proceedings of International Conference on Recent Advances in Neurotraumatology, Gold Coast, Queensland, Australia, 141-144, 1994.
89.Wen-Ta Chiu, Keewhan Choi, Ching-Chang Hung, Chun-Jen shih, and Ronald E. LaPorte: A Briefing of First Intermational Symposium of Epidemiology of Head and Spinal Cord Injury in Developing Countries. Epidemiology of Head and Spinal Injury in Developing Countries and Global SHIP program. International symposium on epidemiology of head & spinal cord injury in developing countries, 1-5, 1994.
90.Wen-Ta Chiu, Ching-Chang Hung, Chun-Jen shih: Epidemiology of head injury in rural Taiwan- a four year survey. Journal of Clinical Neuroscience, 2(3): 210-215, 1995.
91.Wen-Ta Chiu: The Motorcycle Helmet Law in Taiwan, JAMA, 274(12): 941-942, 1995.
92.Wen-Ta Chiu, Kau-Hsun Yeh, YC Li, TH Gan, HY Chen, CC Hung: Traumatic brain injury registry in Taiwan. Neurological Research, 19: 261-264, 1997.
93.Wen-Ta Chiu, Ching-Chang Hung, Liang-Sghong Le, Lieh-Sheng Lin, Chun-Jen Shih, Ronald E. Laporte: Head Injury in Urban and Rural Populations in a Developing Country. Journal of Clinical Neuroscience, 4(4): 469-472, 1997.
94.Wen-Ta Chiu, Jennifer C.Y. Lin : The Impact of Helmet Use and Its Effect on Head Injuries – a Review. New Taipei Journal of Medicine, 1 (2): 63-68, 1999.
95.Wen-Ta Chiu, Chia-Ying Kuo, ching-chang Hung, Marcelo Chen: The effect of the Taiwan Motorcycle Helmet Use Law on Head Injury. American Journal of Public Health, 90(5): 793-796, 2000.
96.Wen-Ta Chiu, Jeffrey Arnold, Yaw-Tang Shih, Kuang-Hua Hsiung, Hsueh-Yun Chi, Chia-Huei Chiu, Wan-Chen Tsai, William Huang: A Survey of International Urban Search and Rescue Teams Following the Ji Ji Earthquake. Disasters, 26(1): 85-94, 2002.
97.Wen-Ta Chiu, Sheng-Jean Huang, Hei-Fen Hwang, Jau-Yih Tsauo, Chun-Fu Chen, Shih-Han Tsai, Mau-Roung Lin (2006) Use of the WHOQOL-BREF for Evaluating Persons with Traumatic Brain Injury. Journal of Neurotrauma 23(11):1609-1620.
98.Wen-Ta Chiu, Tien-Jen Lin, Jia-Wei Lin, Sheng-Jean Huang, Cheng-Kuei Chang, Hsiang-Yin Chen*(2006) Multi-center evaluation of propofol for head-injured patients in Taiwan. Surgical Neurology 66 (Suppl 2): S37-42.
99.Wen-Yin Hsueh, Wen-Ta Chiu, Hui-Wen Lin, Chia-Ying Kuo, Chien-Ten Su: Epidemiologic Study of Head Injury in the Elderly in Taiwan Area. Journal of Taipei Medical College, 24(12): 22-28, 1995.
100.Werner C, Engelhard K. (2007) Pathophysiology of traumatic brain injury. Br J Anaesth. 99(1): 4-9.
101.Yeou-Chih Wang, Dar-Yu Yang, Wen-Ta Chiu, et al.: Epidemiology of Head Injury in Central Taiwan. Epidemiology of Head and Spinal Cord Injury in Developing Countries. International symposium on epidemiology of head & spinal cord injury in developing countries,137-138, 1994.
102.Yildiz S, Aktas S, Cimsit M, Ay H, Toğrol E. (2004) Seizure incidence in 80,000 patient treatments with hyperbaric oxygen. Aviat Space Environ Med. 75(11): 992-994.
103.Yoshida S, Tajika T, Yamasaki N, Tanikawa T, Kitamura K, Kudo K, Lyden P: Dialysis dysequilibrium syndrome in neurosurgical patients. Neurosurgery 1987, 20:716-721.

------------------------------------------------------------------------ 第 2 筆 ---------------------------------------------------------------------
系統識別號 U0007-0502200911080600
論文名稱(中文) 脊病變之細胞再生治療的動物模式及分子造影監測
論文名稱(英文) Cell Regenerative Therapy for Spinal Lesion in Animal Model Monitored by Molecular Imaging
校院名稱 臺北醫學大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Graduate Institute of Clinical Medicine
學年度 97
學期 1
出版年 98
研究生(中文) 羅文政
學號 D102092012
學位類別 博士
語文別 英文
口試日期 2009-01-24
論文頁數 151頁
口試委員 委員-江漢聲
委員-黃文盛
委員-蔡瑞章
委員-邱文達
委員-蔣永孝
共同指導教授-鄧文炳
指導教授-吳志雄
關鍵字(中) 脊病變
細胞再生
動物模式
分子造影監測
關鍵字(英) cell regenerative therapy
spinal lesion
animal model
molecular imaging
學科別分類
中文摘要 脊椎的構造具有包括保護支撐功能的脊椎骨骼、韌帶、關節等,受脊保護的內部構造包括脊髓神經及血管。脊椎骨疾病中,壓迫性骨折常是病患行動困難及疼痛的主要原因,而骨質疏鬆是導致壓迫性骨折最主要因素。另外一個難以治療的脊疾病就是脊髓損傷。雖然每年有許許多多有關脊髓損傷的病理機轉及分子生物學的研究,但是目前仍無法有效的改善病患臨床症狀。因此我們嘗試以NIH3T3細胞治療的作為脊髓損傷治療的另一選擇。NIH3T3具有局部分化的功能且有自體分泌生長因子如GDNF能力。首先我們將NIH3T3植入雙重報告基因,再將它用於動物脊椎疾病的細胞治療技術中,再以分子影像學來直接觀察NIH3T3的作用軌跡。同時也以分子生物學技術來分析此種治療的可能機轉。本論文包含兩主題。第一主題是利用由胚胎纖維母細胞NIH3T3,進行脊髓挫傷後之細胞治療。NIH3T3同時具有能分泌內生性膠質細胞延伸生長因子(GDNF)。第二主題是將去卵巢的SAMP8早老化老鼠骨頭,注入利用富含血小板血漿處置後的胚胎纖維母細胞NIH3T3,引導骨頭新生作用以分子影像來追蹤觀察NIH3T3 遷移的現象,同時以分子生物技術分析體外及體內NIH3T3成骨細胞分化能力及骨化現象。以下兩主題共同特性包括臨床上皆為脊疾病相關的現代疾病,而且使用相同細胞NIH3T3作為細胞治療的細胞來源摘要。同時以分子影像監測NIH3T3遷移能力。並且以分子生物技術分析NIH3T3於體外及體內的分化能力。以下兩主題分別敘述。
第一主題利用具有能分泌內生性膠質細胞衍生生長因子(GDNF)由胚胎纖維母細胞NIH3T3,進行脊髓挫傷後之細胞治療並評估其治療結果。首先在Long-Eions大鼠的第十胸椎脊髓以NYU撞擊機撞擊,已造成急性脊髓損傷。然後再以帶有雙重報告基因的NIH3T3細胞,含有腺嘧啶激酶(Thymidie Kinase)及綠色螢光蛋白(green fluorescent protein)基因,植入第二腰椎脊髓,進而觀察受傷脊髓部分的細胞增生、分化及凋亡預防的情況。另外在活體內以核子醫學或螢光影像追蹤NIH3T3-TG。在平面以螢光影像追蹤顯影發現,NIH3T3-TG能在早期術後二小時,即能發現NIH3T3-TG從植入處第二腰椎往上遷移2公分。而此NIH3T3-TG遷移訊號能藉由核子醫學追蹤達48小時。同時經由免疫組織化學分析,發現無論於活體外或活體內NIH3T3-TG皆能分泌GDNF。在脊髓損傷三週後,植入的NIH3T3-TG不但能從第二腰椎遷移至受傷的第十胸椎,亦能分泌GDNF,產生抗凋亡作用。最後NIH3T3-TG細胞在活體外的細胞分化實驗中,以誘導神經分化的培養液引導,發現其具有與神經細胞型態上及基因上相似的分化潛力。
第二主題是探討骨質疏鬆。骨質疏鬆是現代常見之疾病,骨質疏鬆導致的脊椎壓迫性骨折,在美國每年約增加70萬病例。本研究的目的在於發展一種利用細胞為基礎的治療方法來引導骨頭再生,並以分子影像及免疫生化反應來追蹤驗證。我們利用含螢光報導基因的胚胎纖維母細胞NIH3T3-G,先在體外以富含血小板血漿(PRP)引導分化成骨細胞樣細胞,再將此細胞移植入去卵巢早老化的老鼠(OOX-SAMP8)骨內。結果發現此去卵巢早老化的老鼠能免於骨質疏鬆的發生。從分子影像學及免疫生化學表現發現成骨細胞樣分化的NIH3T3-G細胞能由植入處遷移至遠端的四肢骨骼。以免疫生化分析反應發現遷移處的骨樣細胞同時表現osteopontin及GFP在新生骨上,證明NIH3T3-G細胞移植在去卵巢早老化的老鼠上能重新增加骨小樑生成及骨密度。有趣的是,PRP/NIH3T3-G細胞移植能延長去卵巢早老化老鼠壽命,由以上實驗,我們發現一種深具潛力的療法,可能可以治療老年停經後的骨質疏鬆。另外對於因為先天基因缺陷相對的加速老化的症狀,如Hutehinson-Gilford Pregeria syndrome,此新療法或許能延長壽命。
綜合以上研究, NIH3T3細胞不但具有自己分泌GDNF的能力,且於體內及體外皆具有分化為神經樣細胞的能力。同時於體外經由 PRP引導分化成骨細胞樣細胞。NIH3T3在植入報導基因後,結合分子影像的組合,是一個具有潛在能力的脊髓傷害及骨質疏鬆細胞治療的研究模式。
英文摘要 The spine anatomy includes the supporting, protecting vertebral skeleton, ligament, joint and the internal protected structures, spinal cord and blood vessel. Osteoporotic spinal compression fracture, one of the many diseases concerning the spine, is often caused severe pain on the patients and therefore induced the difficulties of their actions. Spinal cord injury is another main disease of the spine. Though the topic of the mechanisms of spinal cord injury are studied through pathophysiologist and researched through molecular biology every year, but improvement in the patient's clinical symptom is still ineffective at present. Therefore, the therapeutic option is that we try to regard NIH3T3 as progenitor cells for cell therapy in spinal lesion. Differentiating activities and endogenously express glial cell line-derived neurotrophic factor (GDNF) are the two characteristics found in NIH3T3. After the transplant of reporter gene into NIH3T3, it is used for cell therapy of animal's spinal cord injury disease and osteoporosis disease. Then molecule images were used to monitor the NIH3T3 homing activity for cell therapy. So there are two themes included in this thesis. The first is the usage of the NIH3T3 and the assessment of the NIH3T3 cell therapy after spinal cord injury. The second is the utilization of the NIH3T3 as the cell-based bone regeneration approach evaluated through molecular imaging.
The first study is the evaluation of a novel cell-based therapy, the usage of embryonic-derived NIH3T3 cells that endogenously express glial cell line-derived neurotrophic factor (GDNF) for contusion spinal cord injury (SCI). Proliferation, differentiation and apoptosis prevention were examined following the engraftment of NIH3T3 cells into the spinal cords (L2) of Long-Evans rats subjected to acute SCI at T10 vertebral level by NYU impactor. Dual reporter genes were engineered to be contained by the NIH3T3 cells, namely thymidine kinase (T) and enhanced green fluorescence protein (G), hence NIH3T3-TG. They are used for in vivo cell tracking by both nuclear and fluorescence imaging modalities. It is demonstrated that the transplanted NIH3T3-TG cells at L2 vertebral level have the ability to home two centimeters in distance to the injury site as early as 2 h, and the signals persisted for 48 h post SCI through planar and fluorescence imaging. Immunohistochemical analysis both in vitro and in vivo confirmed that the NIH3T3-TG cells express GDNF. Anti-apoptotic effects were provided by GDNF secreting NIH3T3-TG in the injured cord over the period of 3 weeks. The exhibition of both morphological and genetic resemblance of neuronal cells are found in the NIH3T3-TG cells cultured under the neuronal differentiation medium. As appeared in molecular imaging, GDNF-secreting NIH3T3-TG cells are potential therapeutic models for acute spinal cord injury.
The aim of the second study was the development of a cell-based bone regeneration approach. The study was evaluated by molecular imaging and immunohistochemitry. Methods: Platelet-rich plasma medium (PRP) and intrasosseous transplantation into ovariectomized-senescence-accelerated mice (OVX-SAMP8) were used to pre-differentiated genetically modified NIH3T3 embryonic fibroblasts carrying enhanced green fluorescent protein (NIH3T3-G) were into osteoblast-like cells. Results: The development of osteoporosis was prevented by the PRP-conditioned NIH3T3-G (PRP/NIH3T3-G) engraftment. It is demonstrated through molecular imaging and immunohistochemistry the NIH3T3-G cells migrated from the implantation site throughout the skeleton. It is revealed in the site analysis that the co-expression of osteopontin and GFP are found in the newly formed bone tissue, demonstrating that the bone trabecular architecture and mineral density in treated OVX-SAMP8 mice were repaired. Interestingly, the lifespan was significantly prolonged and showed similarities to congenic senescence resistant strain of mice (SAMR1) in the OVX-SAMP8 mice that received PRP/NIH3T3-G transplantation. Conclusion: The potential of this approach to be applied to treat senile postmenopausal osteoporosis and perhaps inborn genetic syndromes associated with accelerated aging such as Hutchinson-Gilford Progeria Syndrome, and for prolongation of life expectancy in general is very high.
論文目次 Table of Contents
Preface I
List of abbreviations IV
Lists of figures VII
摘要 1
Abstract 4
I. Introduction 7
1. Overview 8
Spinal cord injury and pathogenesis 8
GDNF induced spinal cord regeneration and functional preserve 9
Osteoporosis classification 9
Current therapies for osteoporosis 10
Senescence-accelerated mouse and ovariectomy 11
NIH3T3-TG cells therapy in spinal cord regeneration 11
NIH3T3 proliferation and differentiation in platelet-rich plasma 13
2. AIMS OF THIS STUDY 14
II. Study I
A Novel Cell-Based Therapy for Contusion Spinal Cord Injury Using GDNF-Delivering NIH3T3 Cells with Dual Reporter Genes Monitored by Molecular Imaging 15
1. Introduction 16
2. Materials and Methods 21
3. Results 27
4. Discussion 32
5. Conclusion 37
III. Study II
Transplantation of Embryonic Fibroblast treated with Platelet-Rich Plasma Induces Osteogenesis in SAMP8 Mice Monitored by Molecular Imaging 39
1. Introduction 40
2. Materials and Methods 45
3. Results 54
4. Discussion 60
5. Conclusion 67
IV. Conclusion and Perspectives 69
V. Figures 72
VI. References 89
VII. Appendices 99

參考文獻 (1) Fehlings MG, Sekhon LH, Tator C. The role and timing of decompression in acute spinal cord injury: what do we know? What should we do? Spine. 2001;26(24 Suppl):S101-S110.
(2) Crowe MJ, Bresnahan JC, Shuman SL, Masters JN, Beattie MS. Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys. Nat Med. 1997;3(1):73-76.
(3) Lin LF, Doherty DH, Lile JD, Bektesh S, Collins F. GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons. Science. 1993;260(5111):1130-1132.
(4) Arenas E, Trupp M, Akerud P, Ibanez CF. GDNF prevents degeneration and promotes the phenotype of brain noradrenergic neurons in vivo. Neuron. 1995;15(6):1465-1473.
(5) Wang Y, Lin SZ, Chiou AL, Williams LR, Hoffer BJ. Glial cell line-derived neurotrophic factor protects against ischemia-induced injury in the cerebral cortex. J Neurosci. 1997;17(11):4341-4348.
(6) Klocker N, Braunling F, Isenmann S, Bahr M. In vivo neurotrophic effects of GDNF on axotomized retinal ganglion cells. Neuroreport. 1997;8(16):3439-3442.
(7) Molliver DC, Wright DE, Leitner ML et al. IB4-binding DRG neurons switch from NGF to GDNF dependence in early postnatal life. Neuron. 1997;19(4):849-861.
(8) Bennett DL, Michael GJ, Ramachandran N et al. A distinct subgroup of small DRG cells express GDNF receptor components and GDNF is protective for these neurons after nerve injury. J Neurosci. 1998;18(8):3059-3072.
(9) Henderson CE, Phillips HS, Pollock RA et al. GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle. Science. 1994;266(5187):1062-1064.
(10) Blesch A, Tuszynski MH. GDNF gene delivery to injured adult CNS motor neurons promotes axonal growth, expression of the trophic neuropeptide CGRP, and cellular protection. J Comp Neurol. 2001;436(4):399-410.
(11) Zhou L, Shine HD. Neurotrophic factors expressed in both cortex and spinal cord induce axonal plasticity after spinal cord injury. J Neurosci Res. 2003;74(2):221-226.
(12) Moraleda JM, Blanquer M, Bleda P et al. Adult stem cell therapy: dream or reality? Transpl Immunol. 2006;17(1):74-77.
(13) Cummings BJ, Uchida N, Tamaki SJ et al. Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proc Natl Acad Sci U S A. 2005;102(39):14069-14074.
(14) Iwanami A, Kaneko S, Nakamura M et al. Transplantation of human neural stem cells for spinal cord injury in primates. J Neurosci Res. 2005;80(2):182-190.
(15) Watanabe K, Nakamura M, Iwanami A et al. Comparison between fetal spinal-cord- and forebrain-derived neural stem/progenitor cells as a source of transplantation for spinal cord injury. Dev Neurosci. 2004;26(2-4):275-287.
(16) Reier PJ. Cellular transplantation strategies for spinal cord injury and translational neurobiology. NeuroRx. 2004;1(4):424-451.
(17) Ourednik J, Ourednik V. Graft-induced plasticity in the mammalian host CNS. Cell Transplant. 2004;13(3):307-318.
(18) Ourednik V, Ourednik J. Multifaceted dialogue between graft and host in neurotransplantation. J Neurosci Res. 2004;76(2):193-204.
(19) Hung SC, Deng WP, Yang WK et al. Mesenchymal stem cell targeting of microscopic tumors and tumor stroma development monitored by noninvasive in vivo positron emission tomography imaging. Clin Cancer Res. 2005;11(21):7749-7756.
(20) Ma D, Wang X, Han J. NIH 3T3 cells or engineered NIH 3T3 cells stably expressing GDNF can protect primary dopaminergic neurons. Neurol Res. 2000;22(6):538-544.
(21) Rubin H. 'Spontaneous' transformation as aberrant epigenesis. Differentiation. 1993;53(2):123-137.
(22) Tsai MS, Hwang SM, Tsai YL, Cheng FC, Lee JL, Chang YJ. Clonal amniotic fluid-derived stem cells express characteristics of both mesenchymal and neural stem cells. Biol Reprod. 2006;74(3):545-551.
(23) Basso DM, Beattie MS, Bresnahan JC. Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection. Exp Neurol. 1996;139(2):244-256.
(24) Deng WP, Wu CC, Lee CC et al. Serial in vivo imaging of the lung metastases model and gene therapy using HSV1-tk and ganciclovir. J Nucl Med. 2006;47(5):877-884.
(25) Ray P, Bauer E, Iyer M et al. Monitoring gene therapy with reporter gene imaging. Semin Nucl Med. 2001;31(4):312-320.
(26) Hammarberg H, Piehl F, Cullheim S, Fjell J, Hokfelt T, Fried K. GDNF mRNA in Schwann cells and DRG satellite cells after chronic sciatic nerve injury. Neuroreport. 1996;7(4):857-860.
(27) Houenou LJ, Oppenheim RW, Li L, Lo AC, Prevette D. Regulation of spinal motoneuron survival by GDNF during development and following injury. Cell Tissue Res. 1996;286(2):219-223.
(28) Li L, Wu W, Lin LF, Lei M, Oppenheim RW, Houenou LJ. Rescue of adult mouse motoneurons from injury-induced cell death by glial cell line-derived neurotrophic factor. Proc Natl Acad Sci U S A. 1995;92(21):9771-9775.
(29) Shui C, Scutt AM. Mouse embryo-derived NIH3T3 fibroblasts adopt an osteoblast-like phenotype when treated with 1alpha,25-dihydroxyvitamin D(3) and dexamethasone in vitro. J Cell Physiol. 2002;193(2):164-172.
(30) Satake K, Matsuyama Y, Kamiya M et al. Up-regulation of glial cell line-derived neurotrophic factor (GDNF) following traumatic spinal cord injury. Neuroreport. 2000;11(17):3877-3881.
(31) Raisz LG. Pathogenesis of osteoporosis: concepts, conflicts, and prospects. J Clin Invest. 2005; 115:3318-3325.
(32) Reginster JY, Burlet N. Osteoporosis: a still increasing prevalence. Bone. 2006;
38(2 Suppl 1):S4-S9.
(33) Rosen CJ. Clinical practice. Postmenopausal osteoporosis. N Engl J Med. 2005;
353:595-603.
(34) Chapurlat RD, Delmas PD. Drug insight: Bisphosphonates for postmenopausal
osteoporosis. Nat Clin Pract Endocrinol Metab. 2006; 2:211-219.
(35) Whitfield JF. Osteoporosis-treating parathyroid hormone peptides: What are they? What do they do? How might they do it? Curr Opin Investig Drugs. 2006; 7:349359.
(36) Cao JJ, Wronski TJ, Iwaniec U, Phleger L, Kurimoto P, Boudignon B et al. Aging increases stromal/osteoblastic cell-induced osteoclastogenesis and alters the osteoclast precursor pool in the mouse. J Bone Miner Res. 2005; 20:1659-1668.
(37) Chen TL. Inhibition of growth and differentiation of osteoprogenitors in mouse bone marrow stromal cell cultures by increased donor age and glucocorticoid treatment. Bone. 2004; 35:83-95.
(38) Kawaguchi H. Molecular backgrounds of age-related osteoporosis from mouse genetics approaches. Rev Endocr Metab Disord. 2006; 7:17-22.
(39) Labrie JE, III, Borghesi L, Gerstein RM. Bone marrow microenvironmental
changes in aged mice compromise V(D)J recombinase activity and B cell
generation. Semin Immunol. 2005; 17:347-355.
(40) Phillips JE, Guldberg RE, Garcia AJ. Dermal fibroblasts genetically modified to express Runx2/Cbfa1 as a mineralizing cell source for bone tissue engineering. Tissue Eng. 2007; 13:2029-2040.
(41) Lattanzi W, Parrilla C, Fetoni A, Logroscino G, Straface G, Pecorini G et al. Ex vivo-transduced autologous skin fibroblasts expressing human Lim mineralization protein-3 efficiently form new bone in animal models. Gene Ther. 2008; [advance online publication]
(42) Takeda T, Matsushita T, Kurozumi M, Takemura K, Higuchi K, Hosokawa M.
Pathobiology of the senescence-accelerated mouse (SAM). Exp Gerontol. 1997; 32:117-127.
(43) Flood JF, Morley PM, Morley JE. Age-related changes in learning, memory, and lipofuscin as a function of the percentage of SAMP8 genes. Physiol Behav. 1995; 58:819-822.
(44) Morley JE, Kumar VB, Bernardo AE, Farr SA, Uezu K, Tumosa N et al. Beta-
amyloid precursor polypeptide in SAMP8 mice affects learning and memory.
Peptides. 2000; 21:1761-1767.
(45) Shui C, Scutt AM. Mouse embryo-derived NIH3T3 fibroblasts adopt an
osteoblast-like phenotype when treated with 1alpha,25-dihydroxyvitamin D(3)
and dexamethasone in vitro. J Cell Physiol. 2002; 193:164-172.
(46) Li G, Peng H, Corsi K, Usas A, Olshanski A, Huard J. Differential effect of
BMP4 on NIH/3T3 and C2C12 cells: implications for endochondral bone
formation. J Bone Miner Res. 2005; 20:1611-1623.
(47) Chen WH, Lo WC, Lee JJ, Su CH, Lin CT, Liu HY et al. Tissue-engineered
intervertebral disc and chondrogenesis using human nucleus pulposus regulated
through TGF-beta1 in platelet-rich plasma. J Cell Physiol. 2006; 209:744-754.
(48) Liu Y, Kalen A, Risto O, Wahlstrom O. Fibroblast proliferation due to exposure to a platelet concentrate in vitro is pH dependent. Wound Repair Regen. 2002; 10:336-340.
(49) Arpornmaeklong P, Kochel M, Depprich R, Kubler NR, Wurzler KK. Influence of platelet-rich plasma (PRP) on osteogenic differentiation of rat bone marrow stromal cells. An in vitro study. Int J Oral Maxillofac Surg. 2004; 33:60-70.
(50) Tozum TF, Demiralp B. Platelet-rich plasma: a promising innovation in dentistry. J Can Dent Assoc. 2003; 69:664.
(51) Kanno T, Takahashi T, Tsujisawa T, Ariyoshi W, Nishihara T. Platelet-rich
plasma enhances human osteoblast-like cell proliferation and differentiation. J
Oral Maxillofac Surg. 2005; 63:362-369.
(52) Wan DC, Shi YY, Nacamuli RP, Quarto N, Lyons KM, Longaker MT.
Osteogenic differentiation of mouse adipose-derived adult stromal cells requires retinoic acid and bone morphogenetic protein receptor type IB signaling. Proc Natl Acad Sci U S A. 2006; 103:12335-12340.
(53) Takeda T, Hosokawa M, Takeshita S, Irino M, Higuchi K, Matsushita T et al. A new murine model of accelerated senescence. Mech Ageing Dev. 1981; 17:183194.
(54) Jainchill JL, Aaronson SA, Todaro GJ. Murine sarcoma and leukemia viruses:
assay using clonal lines of contact-inhibited mouse cells. J Virol. 1969; 4:549-553.
(55) Celotti F, Colciago A, Negri-Cesi P, Pravettoni A, Zaninetti R, Sacchi MC. Effect of platelet-rich plasma on migration and proliferation of SaOS-2 osteoblasts: role of platelet-derived growth factor and transforming growth factor-beta. Wound Repair Regen. 2006; 14:195-202.
(56) Phillips JE, Hutmacher DW, Guldberg RE, Garcia AJ. Mineralization capacity of Runx2/Cbfa1-genetically engineered fibroblasts is scaffold dependent.
Biomaterials. 2006; 27:5535-5545.
(57) Rutherford RB, Moalli M, Franceschi RT, Wang D, Gu K, Krebsbach PH. Bone morphogenetic protein-transduced human fibroblasts convert to osteoblasts and form bone in vivo. Tissue Eng. 2002; 8:441-452.
(58) Hirata K, Tsukazaki T, Kadowaki A, Furukawa K, Shibata Y, Moriishi T et al.
Transplantation of skin fibroblasts expressing BMP-2 promotes bone repair more effectively than those expressing Runx2. Bone. 2003; 32:502-512.
(59) Krebsbach PH, Gu K, Franceschi RT, Rutherford RB. Gene therapy-directed
osteogenesis: BMP-7-transduced human fibroblasts form bone in vivo. Hum Gene Ther. 2000; 11:1201-1210.
(60) Zhu W, Boachie-Adjei O, Rawlins BA, Frenkel B, Boskey AL, Ivashkiv LB et al. A novel regulatory role for stromal-derived factor-1 signaling in bone
morphogenic protein-2 osteogenic differentiation of mesenchymal C2C12 cells. J Biol Chem. 2007; 282:18676-18685.
(61) Wang Z, Goh J, Das DS, Ge Z, Ouyang H, Chong JS et al. Efficacy of bone
marrow-derived stem cells in strengthening osteoporotic bone in a rabbit model. Tissue Eng. 2006; 12:1753-1761.
(62) Ishihara A, Roy RR, Ohira Y, Kawano F, Nonaka K, Yamamoto K et al. Effects of aging and exercise on density and cross-sectional area of femur in senescence-accelerated mouse prone 6. J Musculoskelet Neuronal Interact. 2003; 3:162-169.
(63) Chen H, Yao XF, Emura S, Shoumura S. Morphological changes of skeletal
muscle, tendon and periosteum in the senescence-accelerated mouse (SAMP6): A murine model for senile osteoporosis. Tissue Cell. 2006; 38:325-335.
(64) Takada K, Inaba M, Ichioka N, Ueda Y, Taira M, Baba S et al. Treatment of
senile osteoporosis in SAMP6 mice by intra-bone marrow injection of allogeneic bone marrow cells. Stem Cells. 2006; 24:399-405.
(65) Brune T, Bonne G, Denecke J, Elcioglu N, Hennekam RC, Marquardt T et al.
Progeria: a new kind of Laminopathy-- report of the First European Symposium on Progeria and creation of EURO-Progeria, a European Consortium on Progeria and related disorders. Pediatr Endocrinol Rev. 2004; 2:39-45.
(66) Conboy IM, Conboy MJ, Wagers AJ, Girma ER, Weissman IL, Rando TA.
Rejuvenation of aged progenitor cells by exposure to a young systemic
environment. Nature. 2005; 433:760-764.

------------------------------------------------------------------------ 第 3 筆 ---------------------------------------------------------------------
系統識別號 U0007-1105200909404600
論文名稱(中文) 尼古丁及其代謝產物藉由和α7尼古丁受器作用而促進結腸癌細胞轉移之機制探討
論文名稱(英文) Nicotine and its metabolite enhance colon cancer cell migration through reaction with α7-nicotinic acetylcholine receptor – Mechanism study
校院名稱 臺北醫學大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Graduate Institute of Clinical Medicine
學年度 97
學期 2
出版年 98
研究生(中文) 魏柏立
學號 D102094006
學位類別 博士
語文別 中文
口試日期 2009-05-06
論文頁數 98頁
口試委員 指導教授-林時宜
委員-林仁混
委員-陳維熊
委員-王應然
委員-吳志雄
委員-何元順
委員-張育嘉
關鍵字(中) 香煙
尼古丁
尼古丁受器
大腸癌
轉移
關鍵字(英) Smoking
NNK
Nicotine
colon cancer
migration
metastasis
學科別分類
中文摘要 抽煙是一個全球性的健康問題,因為它會增加心血管疾病的發生及多種癌症的產生。由於藥物的進步,心血管疾病的治療有明顯地改善,但癌症的治療卻仍有待加強。香煙中含有上百種的致癌物可以破壞細胞中的基因而引發癌症,但更可怕的是菸草中的尼古丁及其衍生物,尼古丁是讓人們對香煙成癮的主要原因而它的多種衍生物例如NNK也是非常強的致癌物。根據流行病學的研究報告顯示長期抽煙會提高大腸直腸癌致死的風險。因為癌細胞轉移是導致病患死亡的主因,而尼古丁及NNK是否會影響結腸癌細胞的轉移目前仍不清楚,因此需要進一步研究。由於尼古丁及NNK可以藉由和α7尼古丁受器結合來發揮其生物功能所以我首先利用定性和定量的PCR來尋找在結腸癌細胞中是否有α7尼古丁受器,再以Trans-well系統及細胞刮傷試驗來研究尼古丁和NNK對結腸癌細胞轉移的影響並觀察和細胞轉移相關的分子改變,最後用抑制劑及小干擾RNA(siRNA)兩種方法來研究接受器在結腸癌細胞轉移的角色。實驗結果發現在HT29及DLD-1兩株結腸癌細胞上都有α7尼古丁受器(α7-nAChR)。然後以致癌物NNK進行第一部份的實驗: 在Trans-well及細胞刮傷試驗中,發現NNK增加了HT29及DLD-1細胞移動的能力。利用抑制劑及小干擾RNA(siRNA)兩種方法了解NNK是藉由α7尼古丁受器的作用而引發了結腸癌細胞中轉錄抑制因子(Snail及ZEB1)的表現增加並進而抑制結腸癌細胞中E-cadherin的轉錄,因此提高了結腸癌細胞的移動能力。接著以尼古丁進行第二部份的實驗: 尼古丁也是藉由α7尼古丁受器的作用而增進結腸癌細胞的移動能力。但異於NNK會改變E-cadherin的表現,尼古丁一方面藉由引發了結腸癌細胞中Fibronectin的轉錄表現增加,另一方面也引發了COX-2的轉錄表現增加,進而讓結腸癌細胞可以利用血管內皮生長因子(VEGF)及其接受器(VEGFR1)產生自我分泌環(autocrine loop)而提高了結腸癌細胞的移動能力。從我們的實驗得知菸草中的致癌物質NNK能透過結腸癌細胞上的α7尼古丁受器而改變E-cadherin轉錄及其轉錄抑制因子(Snail和ZEB1)的表現量來增加結腸癌細胞的轉移能力,而尼古丁則是透過E-cadherin以外的途徑來促進結腸癌細胞轉移。因此讓我們了解要避免抽煙才能遠離大腸直腸癌,而長期抽煙的民眾必須較一般民眾提早開始進行大腸直腸癌篩檢,如果已經罹患大腸直腸癌則應戒菸才能夠讓治療達到最好的效果。
英文摘要 Smoking is a wourdwide health problem. It increased the risk of cardiovascular disease and caused variant cancers. The treatment of cardiovascular disease improved in recent years but the therapy for cancer remains to be improved. There were more than 100 carcinogens within the tobacco and the devastating link between smoking and cancer is nicotine and its metabolites like NNK, which is one of the most potent carcinogens in tobacco. Long-term cigarette smoking increases the risk of colorectal cancer mortality. Because the major cause of cancer death is metastasis, the influence of nicotine and NNK on the migration of colon cancer cells remains to be determined. Since nicotine and NNK exhibited biologic function by receptor binding, receptor for nicotine and NNK in colon cancer cells was identified by PCR and real time PCR. The influence of nicotine and NNK on migration of colon cancer cells was evaluated by trans-well and wound healing assay. The role of receptor for migration was studied by both inhibitor and small interfering RNA (siRNA). The α7 nicotinic acetylcholine receptor (α7-nAChR) was identified in two colon cancer cell lines, HT29 and DLD-1. NNK enhanced HT29 cell migration in both trans-well and wound healing assays. NNK also enhanced DLD-1 cell migration in dose dependent manner. We used inhibitor and siRNA to demonstrate that α7-nAChR mediated NNK-enhanced colon cancer cell migration and down-regulation of E-cadherin were involved in NNK-enhanced migration of colon cancer cells. Furthermore, Snail and ZEB1, two major transcription repressors of E-cadherin in colon cancers, were induced by NNK treatment. Nicotine also enhanced DLD-1 cell migration at concentration of 1 and 10 μM through α7-nAChR. The nicotine-enhanced migration was mediated by induction of another EMT molecule — Fibronectin, but not E-cadherin. Besides, nicotine also increased expression level of COX-2, VEGF and VEGFR1 which might further caused autocrine loop to enhance the colon cancer cell migration. In conclusion, tobacco specific carcinogen, NNK, enhanced colon cancer metastasis through α7-nAChR and E-cadherin — one of the hallmarks of epithelial mesenchymal transition — and its transcription repressors, Snail and ZEB1. Besides, the nicotine also enhanced the colon cancer cell migration through non-E-cadherin pathway. Therefore, smoking should be added into the risk factors of colorectal cancer. For people with long term smoking history, lowering the age may be considered for colorectal cancer screening. The patients with colorectal cancer should get rid of smoking to achieve the optimal therapeutic result.
論文目次 誌 謝 i
縮寫表 ( Abbreviations ) ii
目 錄 ( Contents ) iii
中文摘要 ( Abstract in Chinese ) v
英文摘要 ( Abstract in English ) vii
第一章 緒論 (Introduction) 1
1. 大腸直腸癌的現況 2
2. 香煙與癌症的關係 5
3. 香煙與大腸直腸癌的關係 9
4. 研究動機及重要性 11
5. 研究的假說與特定目的 12
第二章 實驗材料與方法 ( Materials and Methods ) 13
1. 細胞培養(Cell culture)及藥物處理 14
2. 反轉錄-聚合酶連鎖反應 RT-PCR 14
3. 細胞存活率分析(MTT assay) 16
4. 細胞刮傷試驗 (Wound healing assay) 17
5. 細胞移動能力試驗 (Migration assay) 17
6. 小干擾RNA(siRNA)的製備 18
第三章 實驗結果 ( Results ) 19
1. α7尼古丁受器在結腸癌細胞及大腸直腸癌組織中的表現形態 20
2. NNK在結腸癌細胞沒有增生上的效果 20
3. NNK對於結腸癌細胞移動能力的作用 21
4. NNK經由α7尼古丁受器的作用而增强結腸癌細胞移動的能力 22
5. NNK提高了結腸癌細胞中E-cadherin蛋白之轉錄抑制因子(Snail及ZEB1)的表現進而抑制E-cadherin的生成 23
6. 尼古丁經由α7尼古丁受器作用而增强結腸癌細胞移動的能力 25
7. 尼古丁不會影響E-cadherin的轉錄表現 25
8. 尼古丁會增强Fibronectin的轉錄表現 26
9. 尼古丁也會增强COX-2,VEGF和VEGFR1的轉錄表現 26
第四章 討論 ( Discussion ) 28
第五章 結論與展望 (Conclusion and Perspective) 49
第六章 文獻參考 ( References ) 51
第七章 圖表 ( Tables and Figures ) 65
附錄 ( Appendices ) 95
參考文獻 NIH (2006). "NIH State-of-the-Science Conference Statement on Tobacco Use: Prevention, Cessation, and Control." NIH Consens State Sci Statements 23(3): 1-26.
Aigner, K., B. Dampier, et al. (2007). "The transcription factor ZEB1 (deltaEF1) promotes tumour cell dedifferentiation by repressing master regulators of epithelial polarity." Oncogene 26(49): 6979-88.
Andreolas, C., M. Kalogeropoulou, et al. (2008). "Fra-1 regulates vimentin during Ha-RAS-induced epithelial mesenchymal transition in human colon carcinoma cells." Int J Cancer 122(8): 1745-56.
Ansieau, S., J. Bastid, et al. (2008). "Induction of EMT by twist proteins as a collateral effect of tumor-promoting inactivation of premature senescence." Cancer Cell 14(1): 79-89.
Askari, M. D., M. S. Tsao, et al. (2005). "The tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone stimulates proliferation of immortalized human pancreatic duct epithelia through beta-adrenergic transactivation of EGF receptors." J Cancer Res Clin Oncol 131(10): 639-48.
Bianco, R., R. Rosa, et al. (2008). "Vascular endothelial growth factor receptor-1 contributes to resistance to anti-epidermal growth factor receptor drugs in human cancer cells." Clin Cancer Res 14(16): 5069-80.
Bose, C., H. Zhang, et al. (2005). "Activation of p-ERK1/2 by nicotine in pancreatic tumor cell line AR42J: effects on proliferation and secretion." Am J Physiol Gastrointest Liver Physiol 289(5): G926-34.
Buda, A. and M. Pignatelli (2004). "Cytoskeletal network in colon cancer: from genes to clinical application." Int J Biochem Cell Biol 36(5): 759-65.
Calle, E. E., C. Rodriguez, et al. (2003). "Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults." N Engl J Med 348(17): 1625-38.
Campbell, P. T., K. Curtin, et al. (2008). "Mismatch repair polymorphisms and risk of colon cancer, tumor microsatellite instability, and interactions with lifestyle factors." Gut.
Cano, A., M. A. Perez-Moreno, et al. (2000). "The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression." Nat Cell Biol 2(2): 76-83.
Caristi, S., J. L. Galera, et al. (2001). "Estrogens do not modify MAP kinase-dependent nuclear signaling during stimulation of early G(1) progression in human breast cancer cells." Cancer Res 61(17): 6360-6.
Catassi, A., D. Servent, et al. (2008). "Multiple roles of nicotine on cell proliferation and inhibition of apoptosis: implications on lung carcinogenesis." Mutat Res 659(3): 221-31.
Cavallaro, U. and G. Christofori (2004). "Cell adhesion and signalling by cadherins and Ig-CAMs in cancer." Nat Rev Cancer 4(2): 118-32.
Chambers, A. F., A. C. Groom, et al. (2002). "Dissemination and growth of cancer cells in metastatic sites." Nat Rev Cancer 2(8): 563-72.
Chao, A., M. J. Thun, et al. (2000). "Cigarette smoking and colorectal cancer mortality in the cancer prevention study II." J Natl Cancer Inst 92(23): 1888-96.
Chowdhury, P. and K. B. Udupa (2006). "Nicotine as a mitogenic stimulus for pancreatic acinar cell proliferation." World J Gastroenterol 12(46): 7428-32.
Citarda, F., G. Tomaselli, et al. (2001). "Efficacy in standard clinical practice of colonoscopic polypectomy in reducing colorectal cancer incidence." Gut 48(6): 812-5.
Colangelo, L. A., S. M. Gapstur, et al. (2004). "Cigarette smoking and colorectal carcinoma mortality in a cohort with long-term follow-up." Cancer 100(2): 288-93.
Conti-Fine, B. M., D. Navaneetham, et al. (2000). "Neuronal nicotinic receptors in non-neuronal cells: new mediators of tobacco toxicity?" Eur J Pharmacol 393(1-3): 279-94.
Dasgupta, P. and S. P. Chellappan (2006). "Nicotine-mediated cell proliferation and angiogenesis: new twists to an old story." Cell Cycle 5(20): 2324-8.
De Craene, B., B. Gilbert, et al. (2005). "The transcription factor snail induces tumor cell invasion through modulation of the epithelial cell differentiation program." Cancer Res 65(14): 6237-44.
Debruyne, D., M. J. Oliveira, et al. (2006). "Colon cancer cells: pro-invasive signalling." Int J Biochem Cell Biol 38(8): 1231-6.
Desai, D., L. Chang, et al. (1996). "Synthesis and bioassay of 4-ipomeanol analogs as potential chemopreventive agents against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced tumorigenicity in A/J mice." Cancer Lett 108(2): 263-70.
Diergaarde, B., A. Vrieling, et al. (2003). "Cigarette smoking and genetic alterations in sporadic colon carcinomas." Carcinogenesis 24(3): 565-71.
Egleton, R. D., K. C. Brown, et al. (2008). "Nicotinic acetylcholine receptors in cancer: multiple roles in proliferation and inhibition of apoptosis." Trends Pharmacol Sci.
Epstein, R. J. (2007). "VEGF signaling inhibitors: more pro-apoptotic than anti-angiogenic." Cancer Metastasis Rev 26(3-4): 443-52.
Erler, J. T., K. L. Bennewith, et al. (2006). "Lysyl oxidase is essential for hypoxia-induced metastasis." Nature 440(7088): 1222-6.
Friedl, P. and K. Wolf (2003). "Tumour-cell invasion and migration: diversity and escape mechanisms." Nat Rev Cancer 3(5): 362-74.
Giovannucci, E. (2001). "An updated review of the epidemiological evidence that cigarette smoking increases risk of colorectal cancer." Cancer Epidemiol Biomarkers Prev 10(7): 725-31.
Gotti, C. and F. Clementi (2004). "Neuronal nicotinic receptors: from structure to pathology." Prog Neurobiol 74(6): 363-96.
Guaita, S., I. Puig, et al. (2002). "Snail induction of epithelial to mesenchymal transition in tumor cells is accompanied by MUC1 repression and ZEB1 expression." J Biol Chem 277(42): 39209-16.
Hanahan, D. and R. A. Weinberg (2000). "The hallmarks of cancer." Cell 100(1): 57-70.
Hecht, S. S. (1999). "DNA adduct formation from tobacco-specific N-nitrosamines." Mutat Res 424(1-2): 127-42.
Hecht, S. S. (2003). "Tobacco carcinogens, their biomarkers and tobacco-induced cancer." Nat Rev Cancer 3(10): 733-44.
Hicklin, D. J. and L. M. Ellis (2005). "Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis." J Clin Oncol 23(5): 1011-27.
Ho, Y. S., C. H. Chen, et al. (2005). "Tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces cell proliferation in normal human bronchial epithelial cells through NFkappaB activation and cyclin D1 up-regulation." Toxicol Appl Pharmacol 205(2): 133-48.
Hoffmann, D., A. Rivenson, et al. (1996). "The biological significance of tobacco-specific N-nitrosamines: smoking and adenocarcinoma of the lung." Crit Rev Toxicol 26(2): 199-211.
Huang, C. Y., J. H. Chen, et al. (2005). "Regulation of extracellular signal-regulated protein kinase signaling in human osteosarcoma cells stimulated with nicotine." J Periodontal Res 40(2): 176-81.
Jass, J. R. (2002). "Pathogenesis of colorectal cancer." Surg Clin North Am 82(5): 891-904.
Jemal, A., R. Siegel, et al. (2007). "Cancer statistics, 2007." CA Cancer J Clin 57(1): 43-66.
Jin, Z., F. Gao, et al. (2004). "Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone promotes functional cooperation of Bcl2 and c-Myc through phosphorylation in regulating cell survival and proliferation." J Biol Chem 279(38): 40209-19.
Kang, P. and K. K. Svoboda (2003). "Nicotine inhibits palatal fusion and modulates nicotinic receptors and the PI-3 kinase pathway in medial edge epithelia." Orthod Craniofac Res 6(3): 129-42.
Kang, Y. and J. Massague (2004). "Epithelial-mesenchymal transitions: twist in development and metastasis." Cell 118(3): 277-9.
Konyalian, V. R., D. K. Rosing, et al. (2007). "The role of primary tumour resection in patients with stage IV colorectal cancer." Colorectal Dis 9(5): 430-7.
Labat-Robert, J. (2002). "Fibronectin in malignancy." Semin Cancer Biol 12(3): 187-95.
Landais, E., V. El-Khoury, et al. (2005). "Nicotine induces chromatin changes and c-Jun up-regulation in HL-60 leukemia cells." Oncol Rep 14(6): 1553-8.
Lesslie, D. P., J. M. Summy, et al. (2006). "Vascular endothelial growth factor receptor-1 mediates migration of human colorectal carcinoma cells by activation of Src family kinases." Br J Cancer 94(11): 1710-7.
Livant, D. L. (2005). "Targeting invasion induction as a therapeutic strategy for the treatment of cancer." Curr Cancer Drug Targets 5(7): 489-503.
Loeb, L. A. (1991). "Mutator phenotype may be required for multistage carcinogenesis." Cancer Res 51(12): 3075-9.
Luchtenborg, M., M. P. Weijenberg, et al. (2005). "Cigarette smoking and colorectal cancer: APC mutations, hMLH1 expression, and GSTM1 and GSTT1 polymorphisms." Am J Epidemiol 161(9): 806-15.
Mani, S. A., W. Guo, et al. (2008). "The epithelial-mesenchymal transition generates cells with properties of stem cells." Cell 133(4): 704-15.
Mei, J., H. Hu, et al. (2003). "Transformation of non-cancerous human breast epithelial cell line MCF10A by the tobacco-specific carcinogen NNK." Breast Cancer Res Treat 79(1): 95-105.
Mikkelsen, T. S., J. Hanna, et al. (2008). "Dissecting direct reprogramming through integrative genomic analysis." Nature 454(7200): 49-55.
Minna, J. D. (2003). "Nicotine exposure and bronchial epithelial cell nicotinic acetylcholine receptor expression in the pathogenesis of lung cancer." J Clin Invest 111(1): 31-3.
Mousa, S. and S. A. Mousa (2006). "Cellular and molecular mechanisms of nicotine's pro-angiogenesis activity and its potential impact on cancer." J Cell Biochem 97(6): 1370-8.
Natori, T., M. Sata, et al. (2003). "Nicotine enhances neovascularization and promotes tumor growth." Mol Cells 16(2): 143-6.
O'Connell, J. B., M. A. Maggard, et al. (2004). "Colon cancer survival rates with the new American Joint Committee on Cancer sixth edition staging." J Natl Cancer Inst 96(19): 1420-5.
Okuno, K. (2007). "Surgical treatment for digestive cancer. Current issues - colon cancer." Dig Surg 24(2): 108-14.
Pan, H., J. Califano, et al. (2005). "Loss of heterozygosity patterns provide fingerprints for genetic heterogeneity in multistep cancer progression of tobacco smoke-induced non-small cell lung cancer." Cancer Res 65(5): 1664-9.
Peinado, H., D. Olmeda, et al. (2007). "Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype?" Nat Rev Cancer 7(6): 415-28.
Pena, C., J. M. Garcia, et al. (2006). "The expression levels of the transcriptional regulators p300 and CtBP modulate the correlations between SNAIL, ZEB1, E-cadherin and vitamin D receptor in human colon carcinomas." Int J Cancer 119(9): 2098-104.
Pena, C., J. M. Garcia, et al. (2005). "E-cadherin and vitamin D receptor regulation by SNAIL and ZEB1 in colon cancer: clinicopathological correlations." Hum Mol Genet 14(22): 3361-70.
Perez-Moreno, M., C. Jamora, et al. (2003). "Sticky business: orchestrating cellular signals at adherens junctions." Cell 112(4): 535-48.
Perl, A. K., P. Wilgenbus, et al. (1998). "A causal role for E-cadherin in the transition from adenoma to carcinoma." Nature 392(6672): 190-3.
Pfeifer, G. P., M. F. Denissenko, et al. (2002). "Tobacco smoke carcinogens, DNA damage and p53 mutations in smoking-associated cancers." Oncogene 21(48): 7435-51.
Plate, K. (2001). "From angiogenesis to lymphangiogenesis." Nat Med 7(2): 151-2.
Poon, R. T., S. T. Fan, et al. (2001). "Clinical implications of circulating angiogenic factors in cancer patients." J Clin Oncol 19(4): 1207-25.
Potter, J. D. (1999). "Colorectal cancer: molecules and populations." J Natl Cancer Inst 91(11): 916-32.
Reid, M. E., J. R. Marshall, et al. (2003). "Smoking exposure as a risk factor for prevalent and recurrent colorectal adenomas." Cancer Epidemiol Biomarkers Prev 12(10): 1006-11.
Roy, H. K., T. C. Smyrk, et al. (2005). "The transcriptional repressor SNAIL is overexpressed in human colon cancer." Dig Dis Sci 50(1): 42-6.
Ruoslahti, E. (1999). "Fibronectin and its integrin receptors in cancer." Adv Cancer Res 76: 1-20.
Sarebo, M., C. F. Skjelbred, et al. (2006). "Association between cigarette smoking, APC mutations and the risk of developing sporadic colorectal adenomas and carcinomas." BMC Cancer 6: 71.
Schuller, H. M. (2002). "Mechanisms of smoking-related lung and pancreatic adenocarcinoma development." Nat Rev Cancer 2(6): 455-63.
Schuller, H. M. (2007). "Nitrosamines as nicotinic receptor ligands." Life Sci 80(24-25): 2274-80.
Schuller, H. M. and M. Orloff (1998). "Tobacco-specific carcinogenic nitrosamines. Ligands for nicotinic acetylcholine receptors in human lung cancer cells." Biochem Pharmacol 55(9): 1377-84.
Shin, V. Y., W. K. Wu, et al. (2004). "Nicotine promotes gastric tumor growth and neovascularization by activating extracellular signal-regulated kinase and cyclooxygenase-2." Carcinogenesis 25(12): 2487-95.
Shioiri, M., T. Shida, et al. (2006). "Slug expression is an independent prognostic parameter for poor survival in colorectal carcinoma patients." Br J Cancer 94(12): 1816-22.
Siriwardhana, N., S. Choudhary, et al. (2007). "Precancerous model of human breast epithelial cells induced by NNK for prevention." Breast Cancer Res Treat.
Slattery, M. L., K. Curtin, et al. (2000). "Associations between cigarette smoking, lifestyle factors, and microsatellite instability in colon tumors." J Natl Cancer Inst 92(22): 1831-6.
Sowinski, S., C. Jolly, et al. (2008). "Membrane nanotubes physically connect T cells over long distances presenting a novel route for HIV-1 transmission." Nat Cell Biol 10(2): 211-9.
Streit, M., R. Schmidt, et al. (1996). "Adhesion receptors in malignant transformation and dissemination of gastrointestinal tumors." Recent Results Cancer Res 142: 19-50.
Terry, M. B., A. I. Neugut, et al. (2002). "Risk factors for advanced colorectal adenomas: a pooled analysis." Cancer Epidemiol Biomarkers Prev 11(7): 622-9.
Terry, M. B., A. I. Neugut, et al. (2003). "Tobacco, alcohol, and p53 overexpression in early colorectal neoplasia." BMC Cancer 3: 29.
Thiis-Evensen, E., G. S. Hoff, et al. (1999). "Population-based surveillance by colonoscopy: effect on the incidence of colorectal cancer. Telemark Polyp Study I." Scand J Gastroenterol 34(4): 414-20.
Tsurutani, J., S. S. Castillo, et al. (2005). "Tobacco components stimulate Akt-dependent proliferation and NFkappaB-dependent survival in lung cancer cells." Carcinogenesis 26(7): 1182-95.
Vogelstein, B., E. R. Fearon, et al. (1988). "Genetic alterations during colorectal-tumor development." N Engl J Med 319(9): 525-32.
Wang, S. K., P. H. Liang, et al. (2008). "Targeting the carbohydrates on HIV-1: Interaction of oligomannose dendrons with human monoclonal antibody 2G12 and DC-SIGN." Proc Natl Acad Sci U S A 105(10): 3690-5.
West, K. A., J. Brognard, et al. (2003). "Rapid Akt activation by nicotine and a tobacco carcinogen modulates the phenotype of normal human airway epithelial cells." J Clin Invest 111(1): 81-90.
Willett, W. C. (2000). "Diet and cancer." Oncologist 5(5): 393-404.
Winawer, S. J., A. G. Zauber, et al. (1993). "Prevention of colorectal cancer by colonoscopic polypectomy. The National Polyp Study Workgroup." N Engl J Med 329(27): 1977-81.
Wolin, K. Y., I. M. Lee, et al. (2007). "Leisure-time physical activity patterns and risk of colon cancer in women." Int J Cancer 121(12): 2776-81.
Wu, W. K., H. P. Wong, et al. (2005). "4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone from cigarette smoke stimulates colon cancer growth via beta-adrenoceptors." Cancer Res 65(12): 5272-7.
Wynder, E. L. and J. E. Muscat (1995). "The changing epidemiology of smoking and lung cancer histology." Environ Health Perspect 103 Suppl 8: 143-8.
Xu, L. and X. Deng (2004). "Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induces phosphorylation of mu- and m-calpain in association with increased secretion, cell migration, and invasion." J Biol Chem 279(51): 53683-90.
Xu, L. and X. Deng (2006). "Protein kinase Ciota promotes nicotine-induced migration and invasion of cancer cells via phosphorylation of micro- and m-calpains." J Biol Chem 281(7): 4457-66.
Ye, Y. N., E. S. Liu, et al. (2004). "The modulating role of nuclear factor-kappaB in the action of alpha7-nicotinic acetylcholine receptor and cross-talk between 5-lipoxygenase and cyclooxygenase-2 in colon cancer growth induced by 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone." J Pharmacol Exp Ther 311(1): 123-30.
Ye, Y. N., E. S. Liu, et al. (2004). "Nicotine promoted colon cancer growth via epidermal growth factor receptor, c-Src, and 5-lipoxygenase-mediated signal pathway." J Pharmacol Exp Ther 308(1): 66-72.
Ye, Y. N., W. K. Wu, et al. (2005). "A mechanistic study of colon cancer growth promoted by cigarette smoke extract." Eur J Pharmacol 519(1-2): 52-7.
Zhang, Q., X. Tang, et al. (2007). "Nicotine induces hypoxia-inducible factor-1alpha expression in human lung cancer cells via nicotinic acetylcholine receptor-mediated signaling pathways." Clin Cancer Res 13(16): 4686-94.



------------------------------------------------------------------------ 第 4 筆 ---------------------------------------------------------------------
系統識別號 U0007-1107200900471000
論文名稱(中文) 細胞核數目相異之肺靜脈與左心房心肌細胞在電生理與離子通道的表現
論文名稱(英文) Electrical Activity and Channel Expression in Pulmonary Vein and Left Atrium Cardiomyocytes of Various Nucleus Number
校院名稱 臺北醫學大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Graduate Institute of Clinical Medicine
學年度 97
學期 2
出版年 98
研究生(中文) 黃駿豐
學號 M118096002
學位類別 碩士
語文別 中文
口試日期 2009-06-12
論文頁數 59頁
口試委員 委員-陳耀昌
共同指導教授-葉宏一
委員-謝敏雄
委員-陳識中
指導教授-陳亦仁
關鍵字(中) 心房顫動
左心房
肺靜脈
心肌細胞
單核
雙核
關鍵字(英) atrial fibrillation
left atrium
pulmonary vein
cardiomyocyte
mononucleate
binucleate
學科別分類
中文摘要 前言
心房顫動乃是臨床上最常見的心律不整之一,而且會造成嚴重的心臟功能不良以及增加致死率與發病率。過去的研究已知肺靜脈心肌組織是異位節律點的來源,它會引發陣發性心房顫動及異位性左心房心博過速。但何種心肌細胞容易引發心房顫動仍未有定論。
目的
心肌細胞在胚胎發育過程中,會有不同的細胞核數目產生,其機轉尚未完全明瞭。是否不同的細胞核數目有不同的細胞電生理特性,尚未有相關的研究被提出。因此本實驗的目的在探討單雙核心房與肺靜脈心肌細胞的電生理特性與離子通道的差異,與研究不同細胞核數目的心肌細胞在引發心房顫動機轉中所扮演的角色。

材料與方法
年齡約三個月的雄性兔子 (1.5 到 2 公斤,n = 18),將左心房與肺靜脈的心肌細胞分離出後,以DAPI染細胞核,以區分單核或雙核心肌細胞。以全細胞膜電位箝定法與免疫螢光染色去研究單雙核心肌細胞間的電生理特性,以及離子流與離子通道間的差異。

結果
本實驗主要的發現是 (1) 無論是左心房或不具節律性肺靜脈的單核細胞都較雙核細胞的靜止膜電位為正; (2) 具節律性的肺靜脈心肌細胞中,單核細胞較雙核細胞有較高頻率的節律性; (3) 左心房單核與雙核心肌細胞的IK1離子電流密度沒有顯著差異,但肺靜脈單核心肌細胞的Ik1離子電流密度較雙核細胞小,相同的,肺靜脈單核心肌細胞的Kir 2.3螢光密度也較小; (4) 無論左心房或肺靜脈,單核心肌細胞的ICa,L最大離子電流密度都較雙核細胞為大,而左心房與肺靜脈單核心肌細胞的鈣離子濃度變化也較雙核細胞大; (5) 無論是左心房或肺靜脈,單核心肌細胞都較雙核細胞的RyR2螢光密度為大。

結論
本實驗首次證明左心房與肺靜脈單雙核心肌細胞有不同的電生理特性,且其電生理特性由不同的離子流與離子通道密度所決定,但仍有許多離子流與離子通道特性尚待驗證。此外,單雙核心肌細胞對於引發心房顫動的藥物反應是否不同也值得探討,以進一步釐清核數目相異心肌細胞的生理反應與心房顫動的關聯性。
英文摘要 Introduction

Atrial fibrillation (AF) is the most important clinical arrhythmia which induces cardiac dysfunction and increases mortality and morbidity. Pulmonary veins (PVs) were known to be important sources of ectopic beats with the initiation of paroxysmal atrial fibrillation and the foci of ectopic atrial tachycardia. However, the characteristics of arrhythmogenic cardiomyocytes in left atrium (LA) and pulmonary vein have not been identified.

Aim

The purposes of this study were to evaluate the electrophysiological difference and ion channel properties between mononucleated and binucleated cardiomyocytes in LA and PV.

Material and Methods

Male rabbits of 3months old (n=18; 1.5-2 kg) were sacrificed. Isolated LA-PV cardiomyocytes were obtaied by enzyme. Whole-cell patch clamp and immunostaining were used to study the electroactivity and ion channel of DAPI-identified mononucleated and binucleated cardiomyocytes in LA and PV.

Results

Compared to binucleated cardiomyocytes, mononucleated cardiomyocytes (n=10) have more positive resting membrane potential than binucleated myocytes (n=17) in LA (-57.9±1.0 mV versus -62.2±1.2 mV, P<0.05). Similarly, mononucleated cardiomyocytes (n=10) have more positive resting membrane potential than binucleated cardiomyocytes (n=10) in PV (-56.5±1.1 mV versus -64.0±1.6 mV, P<0.05). In pacemaker PV cardiomyocytes, mononucleated myocytes (n=34) have higher frequency of beating rates than binucleated myocytes (n=34) (2.1±0.2 Hz versus 1.3±0.2 Hz, P<0.05).
Mononucleated cardiomyocytes (n=19) have smaller IK1 current density than binucleated cardiomyocytes (n=12) in PV (-2.6±0.2 pA/pF versus -3.5±0.4 pA/pF, P<0.05). Besides, the ICa,L is larger in mononucleated myocytes (n=16) than in binucleated myocytes (n=15) of LA (-14.2±1.3 pA/pF versus -10.9±0.9 pA/pF, P< 0.05). The ICa,L is also larger in mononucleated cardiomyocytes (n=18) than in binucleated cardiomyocytes (n=18) of LA (-9.3±0.7 pA/pF versus -7.0±0.8 pA/pF, P<0.05). In PV,. the RyR2 density of mononucleated myocytes (n=27) is higher than that of binucleated myocytes (n=17) of LA (100.3±4.2 IU/μm2 versus 85.1±3.3 IU/μm2, P<0.05). The RyR2 density of mononucleated cardiomyocytes (n=16) is also higher than that of binucleated cardiomyocytes (n=18) of PV (139.8±5.0 IU/μm2 versus 124.1±5.4 IU/μm2, P<0.05).

Moreover, the mononucleated myocytes (n=20) had a larger [Ca2+]i transient than the binucleated myocytes (n=10) in LA (F-F0/F0, 0.52±0.06 IU versus 0.19±0.05 IU, P<0.05). Similarly, the amplitude of the [Ca2+]i transient of mononucleated cardioimyocytes (n=15) was also larger than that of binucleated cardiomyocytes (n=10) in PV (F-F0/F0, 0.64±0.09 IU versus 0.20±0.03 IU, P<0.05). In addition, the duration of [Ca2+]i transients of mononucleated myocytes (n=20) was longer than that of binucleated myocytes (n=10) in LA (67.9±6.9 ms versus 40.2±3.7 ms, P<0.05). In PV, as compared with binucleated cardiomyocytes (n=10), the mononucleated cardiomyocytes (n=15) also had a longer duration of [Ca2+]i transients (69.2±5.3 ms versus 45.3±5.9 ms, P<0.05)

Conclusions

The study first demonstrate the different electrophysiology characteristics between mononucleated and binucleated cardiomyocytes in LA and PV. Moreover, this study demonstrated the feasibility to examine the response of mononucleated and binucleated cardiomyocytes to drugs that were shown to induce AF.
論文目次 中文摘要------------------------------------------------------------------------------1
英文摘要------------------------------------------------------------------------------3
第一章 緒論--------------------------------------------------------------------------5
第一節 心房顫動-----------------------------------------------------------------5
第二節 肺靜脈心肌細胞的介紹-----------------------------------------------7
第三節 心律不整的介紹-------------------------------------------------------10
第四節 離子通道與離子流的介紹-------------------------------------------12
第二章 研究目的-------------------------------------------------------------------16
第三章 材料與方法----------------------------------------------------------------17
第一節 實驗動物----------------------------------------------------------------17
第二節 左心房-肺靜脈心肌細胞的分離------------------------------------17
第三節 電生理學的研究-------------------------------------------------------18
第四節 免疫螢光染色的研究-------------------------------------------------21
第五節 試劑與抗體-------------------------------------------------------------23
第六節 實驗數據與統計-------------------------------------------------------25
第四章 實驗結果-------------------------------------------------------------------26
第一節 左心房及肺靜脈單雙核心肌細胞電生理特性的比較---------26
第二節 左心房及肺靜脈單雙核心肌細胞各離子流的比較------------27
第三節 左心房及肺靜脈單雙核心肌細胞各離子通道與鈣離子免疫螢光染色的比較 --------------------------------------------------------28
第五章 討論-------------------------------------------------------------------------31
第一節 左心房及肺靜脈單雙核心肌細胞電生理特性與離子流的探討-----------------------------------------------------------------------------------------32
第二節 左心房及肺靜脈單雙核心肌細胞各離子通道與鈣離子流免疫螢光染色的比較--------------------------------------------------------34
第三節 結論與展望--------------------------------------------------------------35
參考文獻-----------------------------------------------------------------------------37
圖--------------------------------------------------------------------------------------45
參考文獻 1. Pai SM and Torres V. Atrial fibrillation: new management strategies. Curr Probl Cardiol 1993;18: 235-300.
2. Wolf PA, Abbott RD, and Kannel WB. Atrial fibrillation: a major contributor to stroke in the elderly. The Framingham Study. Arch Intern Med 1987;147: 1561-1564.
3. Wolf PA, Dawber TR, Thomas HE, Jr., and Kannel WB. Epidemiologic assessment of chronic atrial fibrillation and risk of stroke: the Framingham study. Neurology 1978;28: 973-977.
4. Nattel S. New ideas about atrial fibrillation 50 years on. Nature 2002;415: 219-226.
5. Kourie JI. Interaction of reactive oxygen species with ion transport mechanisms. Am J Physiol 1998;275: C1-24.
6. Garrey WE. Auricular fibrillation. physiol Rev. 1924;4:215-250.
7. Moe GK, Rheinboldt WC, Abildskov JA. A computer model of
atrial fibrillation. Am Heart J. 1964;67:200-220.
8. Bosch RF, Nattel S. Cellular electrophysiology of atrial fibrillation.
Cardiovasc Res. 2002;54:259-269.
9. Allessie MA, Bonke FI, Schopman FJ. Circus movement in rabbit
atrial muscle as a mechanism of tachycardia. III. The "leading
circle" concept: a new model of circus movement in cardiac tissue
without the involvement of an anatomical obstacle. Circ Res.
1977;41:9-18.
10. Allessie M, Ausma J, Schotten U. Electrical, contractile and
structural remodeling during atrial fibrillation. Cardiovasc Res.
2002;54:230-246.
11. Haissaguerre M, Jais P, Shah DC, Gencel L, Pradeau V, Garrigues
S, Chouairi S, Hocini M, Le Metayer P, Roudaut R, Clementy J.
Right and left atrial radiofrequency catheter therapy of paroxysmal
atrial fibrillation. J Cardiovasc Electrophysiol. 1996;7:1132-1144.
12. Haissaguerre M, Jais P, Shah DC, Takahashi A, Hocini M, Quiniou
G, Garrigue S, Le Mouroux A, Le Metayer P, Clementy J.
Spontaneous initiation of atrial fibrillation by ectopic beats
originating in the pulmonary veins. N Engl J Med. 1998;339:
659-666.
13. Nattel S. Therapeutic implications of atrial fibrillation mechanisms:
can mechanistic insights be used to improve AF management?
Cardiovasc Res. 2002;54:347-360.
14. Wijffels MC, Kirchhof CJ, Dorland R, Allessie MA. Atrial
fibrillation begets atrial fibrillation. A study in awake chronically
instrumented goats. Circulation. 1995;92:1954-1968.
15. Cheung DW. Electrical activity of the pulmonary vein and its
interaction with the right atrium in the guinea-pig. J Physiol.
1980;314:445-456.
16. de Bakker JM, Ho SY, Hocini M. Basic and clinical
electrophysiology of pulmonary vein ectopy. Cardiovasc Res.
2002;54:287-294.
17. Verheule S, Wilson EE, Arora R, Engle SK, Scott LR, Olgin JE.
Tissue structure and connexin expression of canine pulmonary
veins. Cardiovasc Res. 2002;55:727-738.
18. Hocini M, Ho SY, Kawara T, Linnenbank AC, Potse M, Shah D,
Jais P, Janse MJ, Haissaguerre M, De Bakker JM. Electrical
conduction in canine pulmonary veins: electrophysiological and
anatomic correlation. Circulation. 2002;105:2442-2448.
19. Spach MS, Barr RC, Jewett PH. Spread of excitation from the
atrium into thoracic veins in human beings and dogs. Am J Cardiol.
1972;30:844-854.
20. Spach MS, Miller WT, III, Dolber PC, Kootsey JM, Sommer JR,
Mosher CE, Jr. The functional role of structural complexities in the
propagation of depolarization in the atrium of the dog. Cardiac
conduction disturbances due to discontinuities of effective axial
resistivity. Circ Res. 1982;50:175-191.
21. Cheung DW. Pulmonary vein as an ectopic focus in digitalis-induced
arrhythmia. Nature. 1981;294:582-584.
22. Masani F. Node-like cells in the myocardial layer of the pulmonary
vein of rats: an ultrastructural study. J Anat. 1986;145:133-142.
23. DeRuiter MC, Gittenberger-De Groot AC, Wenink AC, Poelmann
RE, Mentink MM. In normal development pulmonary veins are
connected to the sinus venosus segment in the left atrium. Anat Rec.
1995;243:84-92.
24. Chen YJ, Chen SA, Chen YC, Yeh HI, Chang MS, Lin CI.
Electrophysiology of single cardiomyocytes isolated from rabbit
pulmonary veins: implication in initiation of focal atrial fibrillation.
Basic Res Cardiol. 2002;97:26-34.
25. Chen YJ, Chen SA, Chen YC, Yeh HI, Chan P, Chang MS, Lin CI.
Effects of rapid atrial pacing on the arrhythmogenic activity of
single cardiomyocytes from pulmonary veins: implication in
initiation of atrial fibrillation. Circulation. 2001;104:2849-2854.
26. Chen SA, Tai CT, Tsai CF, Hsieh MH, Ding YA, Chang MS.
Radiofrequency catheter ablation of atrial fibrillation initiated by
pulmonary vein ectopic beats. J Cardiovasc Electrophysiol. 2000;
11:218-227.
27. Lin WS, Prakash VS, Tai CT, Hsieh MH, Tsai CF, Yu WC, Lin YK,
Ding YA, Chang MS, Chen SA. Pulmonary vein morphology in
patients with paroxysmal atrial fibrillation initiated by ectopic beats
originating from the pulmonary veins: implications for catheter
ablation. Circulation. 2000;101:1274-1281.
28. Wu TJ, Doshi RN, Huang HL, Blanche C, Kass RM, Trento A,
Cheng W, Karagueuzian HS, Peter CT, Chen PS. Simultaneous
biatrial computerized mapping during permanent atrial fibrillation
in patients with organic heart disease. J Cardiovasc Electrophysiol.
2002;13:571-577.
29. Chen MS, Marrouche NF, Khaykin Y, Gillinov AM, Wazni O,
Martin DO, Rossillo A, Verma A, Cummings J, Erciyes D, Saad E,
Bhargava M, Bash D, Schweikert R, Burkhardt D,
Williams-Andrews M, Perez-Lugones A, Abdul-Karim A, Saliba W,
Natale A. Pulmonary vein isolation for the treatment of atrial
fibrillation in patients with impaired systolic function. J Am Coll
Cardiol. 2004;43:1004-1009.
30. Ehrlich JR, Cha TJ, Zhang L, Chartier D, Melnyk P, Hohnloser SH,
Nattel S. Cellular electrophysiology of canine pulmonary vein
cardiomyocytes: action potential and ionic current properties. J
Physiol. 2003;551:801-813.
31. Chen SA, Hsieh MH, Tai CT, Tsai CF, Prakash VS, Yu WC, Hsu
TL, Ding YA, Chang MS. Initiation of atrial fibrillation by ectopic
beats originating from the pulmonary veins: electrophysiological
characteristics, pharmacological responses, and effects of
radiofrequency ablation. Circulation. 1999;100:1879-1886.
32. Chen YJ, Chen SA, Chang MS, Lin CI. Arrhythmogenic activity of
cardiac muscle in pulmonary veins of the dog: implication for the
genesis of atrial fibrillation. Cardiovasc Res. 2000;48:265-273.
33. Chen YC, Chen SA, Chen YJ, Chang MS, Chan P, Lin CI. Effects
of thyroid hormone on the arrhythmogenic activity of pulmonary
vein cardiomyocytes. J Am Coll Cardiol. 2002;39:366-372.
34. Waldo AL and Wit AL. Mechanisms of cardiac arrhythmias. Lancet 1993;341: 1189-1193.
35. Chen YJ, Chen YC, Yeh HI, Lin CI, and Chen SA. Electrophysiology and arrhythmogenic activity of single cardiomyocytes from canine superior vena cava. Circulation 2002;105: 2679-2685.
36. Zeng J and Rudy Y. Early afterdepolarizations in cardiac myocytes: mechanism and rate dependence. Biophys J 1995;68: 949-964.
37. Pogwizd SM and Bers DM. Cellular basis of triggered arrhythmias in heart failure. Trends Cardiovasc Med 2004;14: 61-66.
38. Verkerk AO, Veldkamp MW, Bouman LN, and van Ginneken AC. Calcium-activated Cl(-) current contributes to delayed afterdepolarizations in single Purkinje and ventricular myocytes. Circulation 2000;101:2639-2644.
39. Kleiman RB, and Houser SR. Calcium currents in normal and
hypertrophied isolated feline ventricular myocytes. Am J Physiol 1998;255:H1434-1442.
40. Kucera JP, Rohr S, and Rudy Y. Localization of sodium channels in
intercalated disks modulates cardiac conduction. Circ Res 2002;91:1176-1182.
41. Kuo HC, Cheng CF, Clark RB, Lin JJ, Lin JL, Hoshijima M, Nguyen-Tran VT, Gu Y, Ikeda Y, Chu PH, Ross J, Giles WR, and Chien KR. A defect in the Kv channel-interacting protein 2 (KChIP2) gene leads to a complete loss of I(to) and confers susceptibility to ventricular tachycardia. Cell 2001;107:801-813.
42. Lau CP, Tse HF, and Ayers GM. Defibrillation-guided radiofrequency
ablation of atrial fibrillation secondary to an atrial focus. J Am Coll Cardiol 1999;33:1217-1226.
43. Lebeche D, Kaprielian R, and Hajjar R. Modulation of action potential
duration on myocyte hypertrophic pathways. J Mol Cell Cardiol
2006;40:725-735.
44. Li J, Patel VV, Kostetskii I, Xiong Y, Chu AF, Jacobson JT, Yu C, Morley GE, Molkentin JD, and Radice GL. Cardiac-specific loss of N-cadherin leads to alteration in connexins with conduction slowing and arrhythmogenesis. Circ Res 2005;97: 474-481.
45. DiFrancesco D. The contribution of the ‘pacemaker’ current (if) to generation of spontaneous activity in rabbit sino-atrial node myocytes. J Physiol 1991; 434:23–40.
46. Satoh H. Sino-atrial nodal cells of mammalian hearts: ionic currents and gene expression of pacemaker ionic channels. J Smooth Muscle Res 2003; 39:175–193.
47. Coronado R, Morrissette J, Sukhareva M, Vaughan DN. Structure and function of ryanodine receptors. Am J Physiol Cell Physiol 1994;266: C1485–C1504.
48. Hidalgo C, Bull R, Behrens MI, Donoso P. Redox regulation of RyR-mediated Ca2+ release in muscle and neurons. Biol Res 2004;37: 539–552.
49. Currie S, Smith GL. Enhanced phosphorylation of phospholamban and downregulation of sarco/endoplasmic reticulum Ca2+ ATPase type 2 (SERCA 2) in cardiac sarcoplasmic reticulum from rabbits with heart failure. Cardiovasc Res 1999;41(1): 135.
50. Vangheluwe P, Raeymaekers L, Dode L, Wuytack F. Modulating sarco (endo) plasmic reticulum Ca2+ ATPase 2 (SERCA2) activity: cell biological implications. Cell Calcium 2005;38: 291-302.
51. Jonker SS, Zhang L, Louey S, Giraud GD, Thornburg KL, Faber J. Myocyte enlargement, differentiation, and proliferation kinetics in the fetal sheep heart. J Appl Physiol 2007;102: 1130-1142.
52. Soonpaa MH, Field LJ Survey of studies examining mammalian cardiomyocyte DNA synthesis. Circ Res 1998;83:15–26.
53. MacLennan DH, and Kranias EG. Phospholamban: a crucial regulator
of cardiac contractility. Nat Rev Mol Cell Biol 2003;4:566-577.
54. Masani F. Node-like cells in the myocardial layer of the pulmonary vein of rats: an ultrastructural study. J Anat 1986;145:133-142.

------------------------------------------------------------------------ 第 5 筆 ---------------------------------------------------------------------
系統識別號 U0007-1601200923342900
論文名稱(中文) All trans-RETINOIC ACID 對於鼻咽癌細胞株之生長控制影響以及合併化療藥物治療之效用
論文名稱(英文) Effect of All trans-RETINOIC ACID on the growth of nasopharyngeal cancer cell lines and treatment potential in combined use with cisplatin
校院名稱 臺北醫學大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Graduate Institute of Clinical Medicine
學年度 97
學期 1
出版年 98
研究生(中文) 洪士涵
學號 M102094033
學位類別 碩士
語文別 中文
口試日期 2009-01-07
論文頁數 50頁
口試委員 指導教授-李飛鵬
共同指導教授-吳瑞裕
委員-賴銘堂
委員-黃幸宜
委員-凌慶東
關鍵字(中) 維他命A
視黃質
鼻咽癌
關鍵字(英) Vitamin A
Retinoids
nasopharyngeal cancer
學科別分類
中文摘要 目的:
Retinoids是一群存在於自然界或是人工合成的維生素A的類似物,其中包括了retinol、 isotretinoin、 tretinoin(ATRA)、 retinylpalmitate、 Fenretinide(4-HPR)、 etretinate, arotinoids等等。 此類化合物在許多的動物實驗上,對於許多上皮組織 (包括口腔, 皮膚, 膀胱, 攝護腺和乳腺) 表現出明顯抑制癌化的效果。 近年來各種維他命A的代謝衍生物更被運用到其他類型的癌症上試圖提供更好的腫瘤治療效果。
將ATRA運用在頭頸部鱗狀細胞癌不論是在細胞株的試驗或是動物實驗上已經得到具體的成果,進一步的人體試驗同時也在不同的國家進行當中。 甚至於在一些國家,對於頭頸部鱗狀細胞癌症的治療已經將ATRA納入了原本的化療配方組合之中。 然而,同屬於源自上皮細胞組織的鼻咽癌,卻仍然缺少對於這類促細胞分化藥物的反應研究。
本實驗計畫希望透過ATRA合併使用現有治療鼻咽癌化療藥物Cisplatin,以鼻咽癌細胞株作為實驗材料,試圖找出ATRA對於鼻咽癌細胞的影響,以及合併使用Cisplatin是否具有加成的治療效果。


方法:
實驗材料為兩株本土鼻咽癌細胞株 NPC-TW01以及 NPC-TW04。 各別於不同濃度之ATRA培養下,在光學顯微鏡下觀察細胞型態學的變化,並測量個別之生長曲線。 最後將細胞株分別培養於混有不同濃度的cisplatin、ATRA、以及兩者藥物合併的培養液中,選定6天之生長期,以MTT assay光學方法測光密度OD值來估算細胞的數目。

結果:
NPC-TW01細胞株在單獨使用Cisplatin 0.5μmol/L 開始產生細胞生長抑制,並且隨著加入藥物的濃度增加而有加強。 單獨使用ATRA 1μmol/L生長開始產生抑制,然而對於細胞生長的抑制作用並未能隨著ATRA濃度的增加而加強。 而NPC-TW04細胞株則對個別使用Cisplatin以及ATRA的反應相較之下明顯變差。
在合併使用的效用上,兩者皆有著顯著的加成效果。 NPC-TW01在ATRA在合併較低濃度的cisplatin之下,對細胞生長抑制的作用與單獨使用ATRA並沒有差異。 然而當cisplatin的濃度達到5μM的時候,合併ATRA 1μM比起對照組可以造成吸光值平均下降至23.7%。 比起單獨使用cisplatin 5μM 以及單獨使用ATRA 1μM,細胞抑制的差異皆可達到統計上的意義。 NPCTW04的反應也有類似的結果,當cisplatin的濃度提升到1 μM以上的時候,加入原本合併使用時反應不甚明顯的最低濃度的ATRA 1μM,就可以達到大幅下降吸光值的效果。

結論:
ATRA對於本實驗中的兩株鼻咽癌細胞株皆有抑制生長的作用,此外當合併使用cisplatin時可以於低濃度就達到良好的加成效果,顯示出ATRA作為鼻咽癌合併治療藥物的潛力。 但是由於在合併cisplatin以及ATRA治療下,腫瘤的抑制加成效果須依賴一定的cisplaitn濃度,而且並未能隨同ATRA的濃度增加而更加明顯。 因此除了同時使用cisplain與ATRA預期可以達到相當好的鼻咽癌合併治療效果之外,若能加入經由不同機轉作用的抗癌藥物,應能進一步再提高療效。
英文摘要 Objectives:
Retinoids are a group of naturally existing or synthetic vitamin A derivatives including retinal, isotretinoin, tretinoin(ATRA), retinylpalmitate, Fenretinide (4-HPR), etretinate, arotinoids. In many animal models, these compounds have shown significant anti-neoplastic effect on many epithelial tissues, and more recently they have been applied to other types of cancer in order to obtain better therapeutic effects.
Previous research have already achieve remarkable results in the application of ATRA on human head and neck squamous cell carcinoma using cell line and animal models. In some countries, ATRA was included as a combined chemotherapy regimen. However, related researches about the nasopharyngeal cancer, which are also originated from the epithelial tissue, were still unclear.
The purpose of this study is to understand the effect of ATRA on the nasopharyngeal cancer cell line and evaluate the additional effect when used together with Cisplatin which is currently a standard chemotherapeutic agent for nasopharyngeal carcinoma.

Materials and Methods:
Two NPC cell lines NPC-TW01 and NPC-TW04 were used and observed under optical microscope for morphological changes after treating with different concentrations of ATRA. Growth curves were also determined individually. Finally after six days culturing with various concentration of ATRA alone, cisplatin alone, and combination of both, viable cell numbers were estimated by OD value using MTT assay.

Results:
Cell growth inhibition can be observed on NPC-TW01 starting with Cisplatin 0.5μmol/L, and this effect was further enhanced by increased cisplatin concentration. Growth inhibition also started with ATRA 1μmol/L treated alone, but unlike cisplain, was not enhanced by increased ATRA concentration. Both cisplatin and ATRA showed decreased inhibitory effect on NPC-TW04.
Both cell lines showed remarkable growth inhibition under combined used of cisplatin and ATRA. Under low concentrations of cisplatin, NPC-TW01 cell growth showed no differences between combined use and using ATRA alone. However, when the concentration of cisplatin increased to 5μM, combined use with ATRA 1μM results in decrease in OD value to 23.7% of control group, which is statistically significant comparing to treating with cisplatin and ATRA alone. Similar result was also found on NPC-TW04. When cisplatin level was raised above 1 μM, combined use with low concentration of ATRA showed remarkable decrease in measured OD value.

Conclusion:
Both cell lines used in this study respond to ATRA and results in cell growth inhibition. Low concentration of ATRA is required for synergy effect while combining with cisplatin, implying the potential use for combination therapy regimen. However, the increased cytotoxicity is cisplatin concentration dependent, and also, not enhanced by further increase of ATRA concentration. Therefore, we expect quite effective nasopharyngeal cancer cell control using ATRA and cisplatin together, and possibly even better control effect if other anti-cancer agents using different mechanisms were added together.
論文目次 中文摘要 …………………………………………………… 1
英文摘要 …………………………………………………… 4
第壹章:緒論
壹、 頭頸癌簡介 ……………………………………………………………… 8
貳、 鼻咽癌簡介 ……………………………………………………………… 9
參、All trans-retinoic acid (ATRA) 與 Cisplatin …………………………………… 11
肆、實驗計劃目的 …………………………………………………………15
第貳章:實驗材料及方法
壹、 實驗材料 ………………………………18
貳、 實驗方法 ………………………………19
第參章:實驗結果
壹、 細胞生長曲線測定 ………………………………………………………24
貳、 顯微鏡下觀察細胞加入ATRA之形態學變化 …………………………………24

參、 測量加入不同濃度之ATRA 以及 Cisplatin 對細胞生長抑制的效果
…………………………………………………………………24
第肆章:實驗討論………………………………………………………………30
參考文獻………………………………………………………36
圖表……………………………………………………41

參考文獻 1. Alan G. Kerr, Scott-Brown`s Otolaryngology, 6th edition volume 5 chapter 13.
2. Louis B. Harrison, Head and Neck Cancer, chapter 27 (pp. 639-667)
3. 國家衛生研究院 鼻咽癌之診斷與治療共識。中華民國八十九年八月出版
4. 朱水松,柯政郁,徐茂銘 預防癌症的生力軍-類視黃質(retinoid)
5. Blomhoff R, Green MH, Berg T & Norum KR. Transport and storage of vitamin A. Science 250: 399-404 (1990).
6. Blaner WS & Olson JA. Retinol and retinoic acid metabolism. In: Sporn MB, Roberts AB & Goodman DS ( eds). The retinoids: biology, chemistry and medicine. Raven Press, New York, p 229-255 (1994).
7. Miller WH, Jr. The emerging role of retinoids and retinoic acid metabolism blocking agents in the treatment of cancer. Cancer 83:1471-1482 (1998).
8. Sporn MB, Roberts AB & DeWitt SG. The retinoids: biology, chemistry and medicine. 2nd edition. Raven Press, New York (1994).
9. Peter C. Adamson. All-Trans Retinoic Acid Pharmacology and its impact on the Treatment of Acute Promyelocytic Leukemia The Oncologist, Oct Vol. 1, No.5, 305-314 (1996),
10. Dimberg A, Oberg F. Retinoic acid-induced cell cycle arrest of human myeloid cell lines. Leuk Lymphoma. Oct; 44(10):1641-50. Review (2003).
11. Dimberg A, Bahram F, Karlberg I, Larsson LG, Nilsson K, Oberg F. Retinoic acid-induced cell cycle arrest of human myeloid cell lines is associated with sequential down-regulation of c-Myc and cyclin E and posttranscriptional up-regulation of p27 (Kip1). Blood. Mar 15; 99 (6):2199-206 (2002).
12. Stephen Trzaska (20 Jun ). “Cisplatin” C&EW News 83 (25) (2005).
13. Stordal. B. and Davey. M. Understanding cisplatin resistance using cellular models. IUBMB Life. 59(11) 696-699 (2007).
14. Aebi S, Kroning R, Cenni et al. All-trans retinoic acid enhances cisplatin-induced apoptosis in human ovarian adenocarcinoma and in squamous head and neck cancer cells. Clin. Cancer Res., 3, 2033-2038 (1997).
15. Masuda M, Toh S, Koike K et al. The role of JNK1 and Stat3 in the response of head and neck cancer cell lines to combined treatment with all-trans retinoic acid and 5-fluorouracil. Jpn. J. Cancer Res. 93, 329-339, (2002).
16. Dong M Shin, Bonnie S, Glisson, et al. Phase II and biologic study of interferon alfa, retinoic acid, and cisplatin in advanced squamous skin cancer. J. Clin Oncol, 20:364-370 (2002).
17. Dennis R. Scribner, Doris M. Benbrook. Retinoids enhance cisplatin-based chmoradiation in cervical cancer cells in vitro. Gyn Onco, 85, 223-225 (2002).
18. Shalinsky DR, Bischoff ED, Gregory ML, et al. Enhanced antitumor efficacy of cisplatin in combination with ALRT1057 (9-cis retinoic acid) in human oral squamous carcinoma xenografts in nude mice. Clin Cancer Res. 2:511-20 (1996).
19. Satake K, Takagi E, Ishii S, et al. Anti-tumor effect of vitamin A and D on head and neck squamous cell carcinoma. Auris Nasus Larynx 30:403-412 (2003).
20. Jiang N, Zhan F, Cao L, Yao K, Li G. c-myc gene inactivation during induction of nasopharyngeal carcinoma cells with retinoic acid. Chin Med J (Engl). Sep;113(9):823-6 (2000).
21. Saadeddin A, Torres-Molina F, Carcel-Trullols, et al. Pharmacokinetics of the time-dependent elimination of all-trans-retinoic acid in rats. AAPS PharmSci. 6(1) (2004).
22. Van Vliet, et al. Retinoic Acid Metabolites in Plasma Are Higher after Intake of Liver Paste Compared with a Vitamin A Supplement in Women. J. Nutr. 131:3197-3203 (2001).
23. Yip and Tsao, Regulation of P63 expression in primary and immortalized nasopharyngeal epithelial cells, Int. J. Oncol. 33:713-724 (2008).
24. Leong et al. The p63/p73 network mediates chemosensitivity to cisplatin in a biologically defined subset of primary breast cancers. J Clin Invest. May 1; 117(5): 1370–1380 (2007).
25. Torigoe et al. Cisplatin resistance and Transcription factors, Curr. Med. Chem.-Anti-Cancer Agents, ,5,15-27 (2005).

------------------------------------------------------------------------ 第 6 筆 ---------------------------------------------------------------------
系統識別號 U0007-1607200911003200
論文名稱(中文) 乳癌的Kruppel-like factor表現和臨床表現的關聯性
論文名稱(英文) The Correlation of Expression of Kruppel-like factors (KLF) and the Clinical Manifestations of the Breast Cancer
校院名稱 臺北醫學大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Graduate Institute of Clinical Medicine
學年度 97
學期 2
出版年 98
研究生(中文) 陳志榮
學號 M102095030
學位類別 碩士
語文別 英文
口試日期 2009-06-12
論文頁數 85頁
口試委員 指導教授-陳志榮
共同指導教授-林賜恩
委員-賴文福
委員-賴義雄
委員-陳達人
關鍵字(中) 乳癌
Kruppel-like factor
關鍵字(英) Breast cancer
Kruppel-like factor
學科別分類
中文摘要 乳腺癌是台灣女性因癌症致死的第四位,乳腺癌的發生率有逐年增加和年輕化的趨勢(40歲[含]以下佔29.3%)。近年來,台灣乳腺癌之年齡分佈的狀況和年輕女性所發生之乳腺癌臨床上較具有較侵犯性的表現與西方國家不同。此外,由於乳腺癌本身的異樣性特質,理想的乳腺癌治療方法不僅須依賴傳統的病理組織學、臨床表徵和常用之生物標誌(如ER、PR、HER2/neu),還需探討與發現新的與預後相關的生物因子。由上所述,對台灣婦女乳腺癌的專門研究,成為一個重要的問題。
Kruppel-like factors (KLFs)是一群鋅指樣轉錄因子(Zinc finger like transcription factors),最初被發現和細胞生長的調控有關,目前已知共有超過20種KLFs。最近幾年有越來越多探討KLF在乳腺癌表現的研究,多數研究主要著重於KLF涉及的致癌機轉,而且有數篇研究認為KLF為乳腺癌新的預後因子。在乳腺癌最重要和最被廣泛探討的有KLF4和KLF5,但研究主要局限於西方國家。為了對本地乳腺癌提供更好的治療策略,本研究的目的是探討KLF4和KLF5在台灣婦女乳腺癌的表現情形和與乳腺癌的生物行為相關性。
過去的文獻指出,KLF4同時有抑癌基因和致癌基因的功能,KLF4不僅促進癌細胞的增生,而且還會調節細胞外基質的產生。此外,較侵犯性的臨床行為可能與KLF4在腫瘤細胞內的分布型態有關,在腫瘤細胞有明顯的核表現時,病人預後較差。KLF5也同時具有抑癌基因和致癌基因的特性。KLF5會促進癌細胞增生和轉化,而且具有高表現量的KLF5的乳腺癌病人的預後不良,這現象和HER-2/neu基因及Ki-67的表現量呈正相關性。高表現量的KLF5通常發生在50歲(含)以下的乳腺癌患者身上。
在本研究中,我們使用免疫組織化學方法探討KLF4和KLF5在非腫瘤及腫瘤(含浸潤癌或原位癌)乳腺組織的表現強度和表現型態。並同時統計KLF4和KLF5的表現與乳腺癌組織學特徵、臨床特徵和其他傳統的預後因子的相關性。
本研究共收集了60名乳腺癌患者,其平均年齡為47歲,平均腫瘤大小為2.7公分。臨床表現為第一期的有30%;第二期有43.3%;第三期有21.7%;第四期有5%。經診斷後的平均追蹤時間為27個月(範圍從8至59個月),其中只有一名患者是死於乳腺癌。本研究中90%病例的乳腺癌的組織型態為浸潤性管道癌(Invasive ductal carcinoma, IDC),且66.7%為中度分化。於病灶旁邊管道原位癌(Ductal carcinoma in situ, DCIS)有60%的分化程度為最差等級的。將KLF4和KLF5的免疫組織染色結果與這些病例的臨床表現作關聯性探討時,發現KLF4表現以細胞質和核為主,其中43.3%的病人為腫瘤部分的表現強度比非腫瘤部分強。若腫瘤細胞的KLF4核表現傾向≧25%的病例,有較高的癌症分期(p=0.006),並有較大的腫瘤(最大徑超過2公分,p=0.035)。KLF4的表現也有年齡的相關性,即年齡超過50歲的病例,浸潤癌或原位癌的表現比非腫瘤部分來的更強(p= 0.007),而且,其浸潤性癌的分化也較差(p=0.033)。此外,我們還發現同一病例的浸潤性癌和原位癌之表現有一致性:若原位癌的表現越強,其浸潤性癌的表現也強(p=0.002);核表現的傾向也有一致性(p<0.001)。KLF5表現以細胞質為主,其中58.3%的病人為腫瘤部分(含浸潤癌或原位癌)的表現強度比非腫瘤部分強。就KLF5方面,浸潤性癌若為陰性或弱的細胞質染色時,比較強細胞質表現的乳癌有較好的組織學分化(p=0.035)。另外,同一腫瘤內KLF5的表現在浸潤性癌和原位癌有一致性:若原位癌的表現越強,其浸潤性癌的表現也強(p<0.001);細胞質表現的傾向也具有一致性(p<0.001)。此外,KLF4的表現強度和型態分別和以下因子的表現無相關:動情素接受器(p=0.271和p=0.925),黃體激素接受器(p=0.191和p=0.448),HER-2/neu(p=0.136和p=0.454),p53(p=1.000和p=0.925)和p21 (p=0.572和p=0.367)。KLF5的表現強度和型態分別和以下因子的表現無相關:動情素接受器(p=1.000和p=0.512),黃體激素接受器(p=1.000)和HER-2/neu(p=0.520和p=0.443)。
本研究發現KLF4表現與浸潤性癌的分期,腫瘤大小,病人年紀有正相關性,未能得出和已知文獻中提到KLF4核表現強,其臨床預後較差的結果;另一方面,KLF5表現與浸潤性癌的分化程度有關,我們同時發現到KLF5的核表現主要是局限在非腫瘤性的乳腺組織(16.7%),且沒有任何核染色在原位癌和浸潤性癌發現,已發表的文獻並未提到此現象。雖然,我們目前還不知道這現象代表的生物意義,但這可能代表KLF5有抑癌基因的作用。我們觀察到KLF4和KLF5表現和乳腺癌的臨床表現有關,但其表現與否還無法作為預測乳腺癌的預後和存活率,主要是由於本研究之病例追蹤時間不夠長,無法明確顯示KLF4與KLF5和存活率的相關性,所以精心設計的回顧性研究,配合上長時間的病人追蹤在研究KLF4和KLF5表現與乳腺癌預後及存活率的關聯性是必要的。
英文摘要 Breast cancer is the fourth cause of female cancer deaths in Taiwan with increased incidence and young age tendency (age?T40 years old, 29.3%). In recent years, the distinct age distribution and more aggressive clinical behavior in the young patient are noted in Taiwanese women and this phenomenon is different from that in the Western countries. Besides, due to the heterogeneity of breast cancer, designation of an ideal treatment protocol for breast cancer could not only be based on the traditionally histological, clinical, and biological markers (such as ER, PR and HER-2/neu) but also some new prognostic factors. Therefore, the specific study of breast cancer in Taiwan women becomes an important issue.
Kruppel-like factors (KLF) belong to a group of zinc finger like transcription factors and are involved in regulating cell proliferation. KLFs have more than twenty subtypes. The studies of Kruppel-like factors in breast cancer are increased recently and are mainly focused on their roles in tumorigenesis. The KLFs are considered as new prognostic factors in breast cancers in some studies. Among them, KLF4 and KLF5 are most important and are broadly studied, but most studies are mainly in Western countries. In order to provide better treatment strategies for native breast cancers, the aim of this study is to evaluate the correlation of KLF4 and KLF5 expression with pathologic changes and clinical behaviors of breast cancers in Taiwanese women.
In the literatures, KLF4 has both tumor suppressor gene and oncogene functions. KLF4 can promote the proliferation of cancer cells and also can regulate production of extracellular matrix. More aggressive clinical manifestations may be associated with the cellular location of KLF4 in cancer cells. The patients have poor prognosis when nuclear localization of KLF4 in cancer cells. KLF5 also has both tumor suppressor gene and oncogene functions. KLF5 can facilitate the proliferation and transformation of cancer cells. Increased expression of KLF5 is a poor prognostic factor and is positively correlated with the expression of HER-2/neu and Ki-67 in breast cancer. KLF5 also has increased expression in breast cancer patients younger than 50 years old.
In this study, we used immunohistochemistry method to evaluate both staining intensity and staining pattern of expression of KLF4 and KLF5 in non-tumor and tumor parts (including invasive and in situ cancers) of breast tissues. We also analyzed the associations of expression status of KLF4 and KLF5 with histological features, clinical presentation and other prognostic factors of breast cancer.
We enrolled 60 breast cancer patients with the mean age 47 years old and the mean tumor size was 2.7 cm. The clinical presentation was stage I: 30.0%; stage II: 43.3%; stage III: 21.7%; and stage IV: 5.0%. The follow-up period of these patients ranged from 8 to 59 months (mean 27 months) and only one patient died of disease. Pathologically, most of them were invasive ductal carcinoma (IDC) (90.0%) and showed moderately differentiation (66.7%). The accompanied ductal carcinoma in situ (DCIS), if present, was predominantly highest grade (60.0%). The immunohistochemical study of KLF4 in cancer cells showed cytoplasmic and nuclear expression. The intensity of tumor part was stronger than non-tumor part in 43.3% patients. We evaluated the association of the immunohistochemical results of KLF4 and KLF5 and clinical manifestations of these patients. We found that more KLF4 nuclear expression in tumor cells positively correlated with more advanced stage (p=0.006) and larger size of the tumor (size more than 2 cm in maximal diameter, p=0.035). KLF4 expression was also age-related. KLF4 intensity was stronger in tumor part than non-tumor part in patients older than 50 years old (p=0.007) and, in this setting, the invasive cancer tended to be poorly differentiated (p=0.033). Besides, consistent expression of KLF4 between DCIS and invasive cancers was also found: stronger intensity in DCIS accompanied with stronger intensity in invasive cancers (p=0.002), more predominant nuclear expression in DCIS with more predominant nuclear expression in invasive cancers (p<0.001). The expression of KLF5 in cancer cells was mainly cytoplasmic. The intensity of tumor part was stronger than non- tumor part in 58.3% patients. For KLF5, invasive breast cancers with negative or weak cytoplasmic expression showed better differentiation compared with strong cytoplasmic expression (p=0.035). Consistent expression of KLF5 between DCIS and invasive cancers was also found: stronger intensity in DCIS with stronger intensity in invasive cancers (p<0.001) and more predominant cytoplasmic expression in DCIS with more predominant cytoplasmic expression in invasive cancers (p<0.001). Moreover, there was no association between the following factors and the KLF4 expression intensity and pattern, respectively: ER (p=0.271 and p=0.925), PR (p=0.191 and p=0.448), HER-2/neu (p=0.136 and p=0.454), p53 (p=1.000 and p=0.925), and p21 (p=0.572 and p=0.367). There was also no correlation between the following factors and the KLF5 expression intensity and staining pattern, respectively: ER (p=1.000 and p=0.512), PR (p=1.000 and p=1.000), and HER-2/neu (p=0.520 and p=0.443).
Our study found that KLF4 expression is positive association with tumor stage, tumor size, and age but could not conduct the conclusion that nuclear KLF4 expression was an adverse prognostic factor proposed in the literatures. In the other hand, KLF5 expression was associated with the differentiation of invasive cancers. We also found that KLF5 nuclear localization was mainly restrictedly in non-tumor breast ducts and lobules (16.7%) and loss of nuclear expression in DCIS and invasive cancers, the finding not mentioned in literatures before. Although we didn’t study the biologic function of KLF5, it maybe presented a possible tumor suppressor gene-like function of KLF5. We found that there were associations of KLF4 and KLF5 expressions and clinical manifestations in breast cancers but the expressions of KLF4 and KLF5 were not enough to predict the prognosis and survival rate. The major cause was due to too short follow up period of our patients to exactly evaluate the association of survival rate and expressions of KLF4 and KLF5. Therefore, well-designed retrospective studies with adequate follow up period for studying correlation of expressions of KLF4 and KLF5 and prognosis and survival rate of breast cancers are necessary.
論文目次 致謝 I
目錄 II
縮寫表 (Abbreviations) IV
第一章 中文摘要 (Abstract in Chinese) 1
第二章 英文摘要 (Abstract in English) 4
第三章 緒論 (Introduction) 8
3.1 Breast Cancer in Taiwan 8
3.2 Kruppel-like Factors and Breast Cancer 14
3.3 Kruppel-like Factor 4 (KLF4) and Breast Cancer 16
3.4 Kruppel-like Factor 5 (KLF5) and Breast Cancer 21
3.5 Aims of Our Study 22
第四章 研究方法與材料 (Materials and Methods) 24
4.1 Study Design and Patient Selection 24
4.2 Pathological Evaluation 24
4.3 Immunohistochemistry 25
4.3.1 Immunohistochemistry of KLF4 in Breast
Cancers 27
4.3.2 Immunohistochemistry of KLF5 in Breast
Cancers 28
4.3.3 Immunohistochemistry of p53 and p21 in
Breast Cancers 28
4.3.4 Immunohistochemistry of ER, PR, and
HER-2/neu in Breast Cancers 28
4.4 Statistical Analysis 30
第五章 實驗結果 (Results) 31
5.1 Clinical Characteristics of the Patients 31
5.2 Pathological Evaluation of Breast Cancers 31
5.3 Immunohistochemical Results of KLF4, KLF5, p53,
and p21 in Breast Cancers 32
5.3.1 Immunohistochemical Results of KLF4 in
Breast Cancers 33
5.3.2 Immunohistochemical Results of KLF5 in
Breast Cancers 34
5.3.3 Immunohistochemical Results of p53 and p21
in Breast Cancers 35
5.4 Correlation of Clinical Features, Histological
Pictures, and Immunohistochemical Results of
KLF4, KLF5, p53, p21, ER, PR, and HER2/neu in
Breast Cancers 36
第六章 討論 (Discussion) 39
第七章 結論與展望 (Conclusion and Perspectives) 49
第八章 參考文獻 (References) 50
第九章 圖表 (Tables and Figures) 56
參考文獻 Beckers J, Herrmann F, Rieger S, Drobyshev AL, Horsch M, Hrabé de Angelis M, Seliger B. Identification and validation of novel ERBB2 (HER2, NEU) targets including genes involved in angiogenesis. Int J Cancer 2005 Apr 20; 114(4):590-7.
Bertheau P, Espié M, Turpin E, Lehmann J, Plassa LF, Varna M, Janin A, de Thé H. TP53 status and response to chemotherapy in breast cancer. Pathobiology 2008; 75(2):132-9.
Black AR, Black JD, Azizkhan-Clifford J.Sp1 and krüppel-like factor family of transcription factors in cell growth regulation and cancer. J Cell Physiol 2001 Aug; 188(2):143-60.
Chen C, Bhalala HV, Qiao H, Dong JT.A possible tumor suppressor role of the KLF5 transcription factor in human breast cancer. Oncogene 2002 Sep 26; 21(43):6567-72.
Chen C, Zhou Y, Zhou Z, Sun X, Otto KB, Uht RM, Dong JT. Regulation of KLF5 involves the Sp1 transcription factor in human epithelial cells. Gene 2004 Apr 14; 330:133-42.
Chen C, Sun X, Guo P, Dong XY, Sethi P, Cheng X, Zhou J, Ling J, Simons JW, Lingrel JB, Dong JT. Human Kruppel-like factor 5 is a target of the E3 ubiquitin ligase WWP1 for proteolysis in epithelial cells. J Biol Chem 2005 Dec 16; 280(50):41553-61.
Chen C, Zhou Y, Dong JT. KLF5: Kruppel-like factor 5 (intestinal). Atlas Genet Cytogenet Oncol Haematol October 2006.
(URL: http://AtlasGeneticsOncology.org/Genes/KLF5ID41074ch13q21.html)
Cheng SH, Tsou MH, Liu MC, Jian JJ, Cheng JC, Leu SY, Hsieh CY, Huang AT. Unique features of breast cancer in Taiwan. Breast Cancer Res Treat 2000 Oct; 63(3):213-23.
Clarke ND, Berg JM. Zinc fingers in Caenorhabditis elegans: finding families and probing pathways. Science 1998 Dec 11; 282(5396):2018-22.
Dang DT, Pevsner J, Yang VW. The biology of the mammalian Krüppel-like family of transcription factors .Int J Biochem Cell Biol 2000; 32(11-12):1103–21.
Elledge RM, Allred DC. Prognostic and predictive value of p53 and p21 in breast cancer. Breast Cancer Res Treat 1998; 52(1-3):79-98.
Faratian D, Bartlett J. Predictive markers in breast cancer – the future. Histopathology 2008; 52: 91–8.
Foster KW, Ren S, Louro ID, Lobo-Ruppert SM, McKie-Bell P, Grizzle W, Hayes MR, Broker TR, Chow LT, Ruppert JM. Oncogene expression cloning by retroviral transduction of adenovirus E1A-immortalized rat kidney RK3E cells: transformation of a host with epithelial features by c-MYC and the zinc finger protein GKLF. Cell Growth Differ 1999 Jun; 10(6):423-34.
Foster KW, Frost AR, McKie-Bell P, Lin CY, Engler JA, Grizzle WE, Ruppert JM. Increase of GKLF messenger RNA and protein expression during progression of breast cancer. Cancer Res 2000 Nov 15; 60(22):6488-95.
Ghaleb AM, Nandan MO, Chanchevalap S, Dalton WB, Hisamuddin IM, Yang VW. Krüppel-like factors 4 and 5: the yin and yang regulators of cellular proliferation. Cell Res 2005 Feb; 15(2):92-6.
Graham JD, Yager ML, Hill HD, Byth K, O'Neill GM, Clarke CL. Altered progesterone receptor isoform expression remodels progestin responsiveness of breast cancer cells. Mol Endocrinol 2005 Nov; 19(11):2713-35.
Guo H, Lin Y, Zhang H, Liu J, Zhang N, Li Y, Kong D, Tang Q, Ma D. Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells. BMC Mol Biol 2007 Dec 3; 8:110.
Kikuchi A. Regulation of beta-catenin signaling in the Wnt pathway. Biochem Biophys Res Commun 2000 Feb 16; 268(2):243-8.
Kumar V, Abbas A, Fausto N. Robbins and Cotran. Pathologic basis of disease, 7th edition, Philadelphia: Elsevier Saunders. 2005; pp:1120-52.
Miller KA, Eklund EA, Peddinghaus ML, Cao Z, Fernandes N, Turk PW, Thimmapaya B, Weitzman SA. Kruppel-like factor 4 regulates laminin alpha 3A expression in mammary epithelial cells. J Biol Chem 2001 Nov 16; 276(46):42863-8.
Pandya AY, Talley LI, Frost AR, Fitzgerald TJ, Trivedi V, Chakravarthy M, Chhieng DC, Grizzle WE, Engler JA, Krontiras H, Bland KI, LoBuglio AF, Lobo-Ruppert SM, Ruppert JM. Nuclear localization of KLF4 is associated with an aggressive phenotype in early-stage breast cancer. Clin Cancer Res 2004 Apr 15; 10(8):2709-19.
Payne SJ, Bowen RL, Jones JL, Wells CA. Predictive markers in breast cancer-the present. Histopathology 2008; 52:82-90.
Rayala SK, Molli PR, Kumar R. Nuclear p21-activated kinase 1 in breast cancer packs off tamoxifen sensitivity. Cancer Res 2006 Jun 15; 66(12):5985-8.
Rosen PP. Rosen’s Breast Pathology. In: Invasive duct carcinoma: assessment of Prognosis, morphologic prognostic markers, and tumor growth rate. Philadelphia: Lippincott Williams & Wilkins; 2009; 358-404.
Rowland BD, Bernards R, Peeper DS. The KLF4 tumor suppressor is a transcriptional repressor of p53 that acts as a context-dependent oncogene. Nat Cell Biol 2005 Nov; 7(11):1074-82.
Rowland BD, Peeper DS. KLF4, p21 and context-dependent opposing forces in cancer. Nat Rev Cancer 2006 Jan; 6(1):11-23.
Shen YC, Chang CJ, Hsu C, Cheng CC, Chiu CF, Cheng AL. Significant difference in the trends of female breast cancer incidence between Taiwanese and Caucasian Americans: implications from age-period-cohort analysis. Cancer Epidemiol Biomarkers Prev 2005 Aug; 14(8):1986-90.
Schnitt SJ, Collins LC. Biopsy interpretation of the breast. In: Invasive breast cancer. Philadelphia: Lippincott Williams & Wilkins; 2009; 249-322.
Staalesen V, Knappskog S, Chrisanthar R, Nordgard SH, Løkkevik E, Anker G, Ostenstad B, Lundgren S, Risberg T, Mjaaland I, Gram IT, Kristensen VN, Børresen-Dale AL, Lillehaug JR, Lønning PE. The novel p21 polymorphism p21G251A is associated with locally advanced breast cancer. Clin Cancer Res 2006 Oct 15; 12(20 Pt 1):6000-4.
Tavassoli FA, Peter Devilee. World health organization classification of Tumours. Pathology& genetics. Tumours of the breast and female genital organs. Lyon: IARC Press; 2003; 15.
Talley L, Chieng DC, Bell WC, Grizzle WE, Frost AR. Immunohistochemical detection of EGFR, p185 (erbB-2), Bcl-2 and p53 in breast carcinomas in pre-menopausal and post-menopausal women. Biotech Histochem 2008 Feb; 83(1):5-14.
Tong D, Czerwenka K, Heinze G, Ryffel M, Schuster E, Witt A, Leodolter S, Zeillinger R. Expression of KLF5 is a prognostic factor for disease-free survival and overall survival in patients with breast cancer. Clin Cancer Res 2006 Apr 15; 12(8):2442-8.
Turner J, Crossley M. Mammalian Krüppel-like transcription factors: more than just a pretty finger. Trends Biochem Sci 1999 Jun; 24(6):236-40.
Vannini I, Zoli W, Tesei A, Rosetti M, Sansone P, Storci G, Passardi A, Massa I, Ricci M, Gusolfino D, Fabbri F, Ulivi P, Brigliadori G, Amadori D, Bonafe M. Role of p53 codon 72 arginine allele in cell survival in vitro and in the clinical outcome of patients with advanced breast cancer. Tumour Biol 2008; 29(3):145-51.
Wang X, Zheng M, Liu G, Xia W, McKeown-Longo PJ, Hung MC, Zhao J. Krüppel-like factor 8 induces epithelial to mesenchymal transition and epithelial cell invasion. Cancer Res 2007 Aug 1; 67(15):7184-93.
Wei X, Xu H, Kufe D.Human mucin 1 oncoprotein represses transcription of the p53 tumor suppressor gene. Cancer Res 2007 Feb 15; 67(4):1853-8.
Yancy HF, Mason JA, Peters S, Thompson CE 3rd, Littleton GK, Jett M, Day AA. Metastatic progression and gene expression between breast cancer cell lines from African American and Caucasian women. J Carcinog 2007 May 1; 6:8.
Zhang L, Wali A, Ramana CV, Rishi AK. Cell growth inhibition by okadaic acid involves gut-enriched Kruppel-like factor mediated enhanced expression of c-Myc. Cancer Res.2007 Nov 1; 67(21):10198-206.
Zhao Y, Hamza MS, Leong HS, Lim CB, Pan YF, Cheung E, Soo KC, Iyer NG. Kruppel-like factor 5 modulates p53-independent apoptosis through Pim1 survival kinase in cancer cells. Oncogene 2008 Jan 3; 27(1):1-8.

------------------------------------------------------------------------ 第 7 筆 ---------------------------------------------------------------------
系統識別號 U0007-1607201015575000
論文名稱(中文) 白藜蘆醇對高糖引起小老鼠間質細胞生長抑制現象的作用
論文名稱(英文) The Effects of Resveratrol on High Glucose-Induced Growth Arrest in Mouse Mesangial Cells
校院名稱 臺北醫學大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Graduate Institute of Clinical Medicine
學年度 98
學期 2
出版年 99
研究生(中文) 陳怡安
學號 M118097002
學位類別 碩士
語文別 中文
口試日期 2010-06-10
論文頁數 72頁
口試委員 指導教授-林裕峰
委員-簡志誠
委員-許永和
關鍵字(中) 白藜蘆醇
高糖環境
抗氧化
凋亡
關鍵字(英) Resveratrol
high glucose
antioxidant
apoptosis
學科別分類
中文摘要 背景:糖尿病腎病變是造成腎衰竭最主要的原因之一,血糖的控制不佳會增加心血管疾病的發生率。慢性的高糖環境下會對腎臟細胞,形成莫大的氧化壓力,氧化產物的過度製造增加,導致腎臟間質細胞的生長抑制及凋亡。我們評估白藜蘆醇這一個多酚構造、多功能的植物天然抗毒素是否可經由抗氧化、抗發炎的過程,減少小鼠腎臟間質細胞暴露在高糖環境下生長抑制及凋亡的現象。研究方法與材料:小鼠腎臟間質細胞分別被培養在含有6.25mM 較低糖分,及35mM 較高糖分環境下,和加入白藜蘆醇做治療。藉由顯微鏡、MTT test觀察其生長狀況。H2-DCFDA偵測細胞內過氧化物質含量。細胞的凋亡判定是運用流式細胞儀和Annexin-V以及propidium iodine的染色。藉由反轉錄聚合脢連鎖反應實驗測量TGFβ及HO-1含量。以及使用ELISA kit 定量 caspsae3。結果:高糖環境會有抑制間質細胞生長、造成細胞內ROS 上升(約增加~70 %;p < 0.05)及促進細胞凋亡現象(增加約1.5~1.8倍;p < 0.05) 。這些反應可因加入合理的生理劑量內的白藜蘆醇(1-2.5μM)而減少。此外也可以下降TGF-β,誘導HO-1酵素生成。可阻斷caspase 3 活性在高糖環境下(p < 0.05)。結論:我們證實了白藜蘆醇經由抗氧化過程可以減緩小鼠腎臟間質細胞暴露在高糖環境下凋亡的現象,而可以改善間質細胞數目的平衡。
英文摘要 Background: Diabetic nephropathy is one major cause of renal failure, and poor glycemic control will increase cardiovascular incidence. Chronic high glucose condition induced oxidative stress, increased reactive oxygen species (ROS) amount, and involves kidney mesangial cell growth arrest and apoptosis pathway. We investigated the therapeutic effect of resveratrol, a polyphenolic compound, with multi-function nature phytoalexin may attenuate high glucose induced cell arrest and apoptosis changes in mouse mesangial cell via anti-oxidant and anti-inflammation proprieties. Material and methods: Mouse mesangial cell were cultured in 6.25mM and 35mM glucose, and add resveratrol in high glucose treated group. Observe cell morphology and viability by microscopy and MTT test. Intracellular ROS generation was assayed by H2-DCFDA. Cell apoptosis amount was determined by flow cytometry via Annexin-V and propidium iodine staining. TGF and HO-1 gene expression was measured by reveres transcriptase- polymerase chain reaction. And caspase 3 activity was quantified by ELISA. Result: High glucose depressed cell growth, increases cellular ROS (~70 %; p < 0.05), and enhanced cell apoptosis (1.5~1.8 fold increase; p < 0.05); these reaction could be attenuated by physiological resveratrol concentration 1-2.5μM. And also noted decreased TGF-β and mediated HO-1 gene expression. Caspase 3 activity was also blocked (p < 0.05). Conclusion: Our findings indicate that resveratrol provides antioxidant effect against high glucose induced cell apoptosis, and may further regulate mesangial cell numbers.
論文目次 中文摘要 (Abstract in Chinese) ----------------------- i
英文摘要 (Abstract in English) ----------------------- ii
第一章 緒論 (Introduction) -----------------------1
第一節 腎臟的構造及糖尿病腎病變的形成--------------- 4
第二節 高糖(High glucose)造成細胞的凋亡( apoptosis)及死亡(death ) -----------------------5
第三節 Transforming growth factor-beta(TGF-β)----------------------- 8
第四節 血红素氧合酶1 (HO-1) ----------------------- 10
第五節 白藜蘆醇(Resveratrol)的藥理作用及臨床應----------------------12
第六節 抗氧化物在腎臟的運用----------------------- 15
第七節 研究目的 ----------------------- 16
第二章 研究方法與材料 (Materials and Methods) ----------------------- 17
第三章 研究結果 (Results)
1.Effect of high glucose on mouse mesangial cell viability ------------------- 26
2.Effects of different dosage resveratrol on mouse mesangial cell viability -----------------------27
3.Effect of resveratrol on high glucose-induced oxidative stress in mouse mesangial cell -----------------------28
4.Effect of resveratrol on high glucose-induced cell apoptosis in mouse mesangial cell-----------------------28
5.Effectof resveratrol on high glucose-induced TGF-β in mouse mesangial cell-----------------------29
6.Effect.of resveratrol mediated HO-1 expression on high glucose treatment in mouse mesangial cell-----------------------30
7.Effect of resveratrol on high glucose-induced caspase-3 activation in mouse mesangial cell-----------------------30
第四章 討論 (Discussion) -----------------------31
第五章 結論與展望 (Conclusion and Perspective) -----------------------45
參考文獻 (References) ----------------------- 46
圖 (Figures) ----------------------- 55
參考文獻 Ayo SH, Radnik RA, Glass WF, et al. Increased extracellular matrix synthesis and mRNA in mesangial cells grown in high glucose medium. Am J Physiol
1991;260:F185–F191.

Agarwal A, Balla J, Alam J ,et al. Induction of heme oxygenase in toxic
renal injury: a protective role in cisplatin nephrotoxicity in the rat. Kidney
Int 1995; 48: 1298–1307.

Abraham NG, Drummond GS, Lutton JD, et al. The biological significance
and physiological role of heme oxygenase. Cell Physiol Biochem 1996; 6:
129–168.

Aizawa T, Ishizaka N, Taguchi J ,et al. Heme oxygenase-1 is upregulated in the kidney of angiotensin II-induced hypertensive rats: possible role in renoprotection. Hypertension 2000; 35: 800–806.

Avihingsanon Y, Ma N, Csizmadia E,et al. Expression of protective genes in human renal allografts: a regulatory response to injury associated with graft rejection. Transplantation 2002; 73: 1079–1085.

Allen DA, Yaqoob MM, Harwood SM: Mechanisms of high glucose-induced apoptosis and its relationship to diabetic complications.J Nutr Biochem 2005; 16: 705–713.

Aziz MH., Nihal M., Fu VX., et al: Resveratrol-caused apoptosis of human prostate carcinoma LNCaP cells is mediated via modulation of phosphatidylinositol 3-kinase/Akt pathway and Bcl-2 family proteins. Mol. Cancer Ther.2006; 5: 1335–1341

Bertelli A, Bertelli AA,Gozzini A, et al. Plasma and tissue resveratrol concentrations and pharmacological activity. Drugs Exp Clin Res 1998; 24:133-138.

Bertelli AA, Ferrara F, Diana G, et al. Resveratrol, a natural stilbene in grapes and wine, enhances intraphagocytosis in human promonocytes: a co-factor in antiinflammatory and anticancer chemopreventive activity. Int J Tissue React 1999;21:93–104.

Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 2001; 414:813– 20.

Bonnefont-Rousselot.Glucose and reactive oxygen species.Curr. Opin. Clin. Nutr. Metab. Care 2002;5: 561–568.

Bamri-Fzzine S, Zhu AO, Londono I ,et al. Apoptosis of tubular epithelial cells in glycogen nephrosis during diabetes. Lab Invest 2003 83: 1069–1080

Chase HP, Jackson WE, Hoops SL, et al. Glucose control and the renal and retinal complications of insulin dependant diabetes. J Am Med Assoc 1989;261: 1155–1160.

Chen S, Hong SW, Iglesias DL ,et al. The key role of the transforming growth factor-β system in the pathogenesis of diabetic nephropathy. Ren Fail 2001; 23: 471–481

Catherwood MA, Powell LA, Anderson P, et al. Glucose-induced oxidative stress in mesangial cells.Kidney Int 2002;61:599– 608.

Cichewicz RH, Kouzi SA.Resveratrol oligomers: structure, chemistry and biological activity, in: A.U. Rahman (Ed.), Studies in Natural Products Chemistry,vol. 26, Elsevier BV, Oxford, UK, 2002, pp. 507–579.

Chen S, Hoffman BB, Lee J, et al. Cultured tubule cells from TGF-β1 null mice exhibit impaired hypertrophy and fibronectin expression in high glucose .Kidney Int 2004; 65: 1191–1204.

Cohen, H. Y., Miller, C., Bitterman, K. J,et al. Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase. Science. 2004;305:
390–392.

Crowell JA, Korytko PJ, Morrissey RL,et al. Resveratrol-Associated Renal Toxicity. Toxico. Sciences 2004;82: 614–619.

Chan WH. Effect of Resveratrol on High Glucose-Induced Stress in Human Leukemia K562 Cells. J. Cell. Biochem. 2005; 94: 1267–1279..

Das DK, Sato M, Ray PS, et al. Cardioprotection of red wine: role of polyphenolic antioxidants. Drugs Under Experimental and Clinical Research 1999;25: 115– 120
Dworkin LD, Ichikawa I, Brenner BM. Hormonal modulation of glomerular function. Am J Physiol 1983;244:F95–F104.

Frankel EN, Waterhouse AL, Kinsella JE. Inhibition of human LDL oxidation by resveratrol. Lancet 1993; 341:1103–1104.

Fremont L. Biological effects of resveratrol. Life Sci 2000;66: 663–73.

Friedlander RM: Apoptosis and caspases in neurodegenerative diseases. N Engl J Med 2003; 348: 1365–1375.

Fulda, S., and Debatin, K. M. Resveratrol modulation of signal transduction in apoptosis and cell survival: a mini-review. Cancer Detect. Prev. 2006; 30: 217–223.

Green DR, Reed JC: Mitochondria and apoptosis. Science 1998; 281: 1309–1312.

Gagliardini G, Benigni A.Role of anti-TGF-β antibodies in the treatment of renal injury Cytokines and Growth factor 2006;16: 89-96.

Goodman AI, Olszanecki R, Yang LM, et al. Heme oxygenase-1 protects against radiocontrast-induced acute kidney injury by regulating antiapoptotic proteins. Kidney Int 2007; 72: 945–953.

Henry DN, Busik JV, Brosius FC, et al. Glucose transporters control gene expression of aldose reductase, PKC and GLUT1 in mesangial cell in vitro. Am J Physiol 1999;277:F97–F104.

Haugen EN, Croatt AJ, Nath KA. Angiotensin II induces renal oxidant stress in vivo and heme oxygenase-1 in vivo and in vitro. Kidney Int 2000; 58: 144–152

Hill-Kapturczak N, Truong L, Thamilselvan V, et al. Smad7-dependent regulation of heme oxygenase-1 by transforming growth factor-b in human renal epithelial cells. J Biol Chem 2000; 275: 40904–40909.

Hong SW, Isono M, Chen S, et al. Increased glomerular and tubular expression of transforming growth factor-β1, its type II receptor, and activation of the Smad signaling pathway in the db/db mouse. Am J Pathol 2001; 158: 1653–1663

Howitz KT,Bitterman KJ,Cohen HY et al. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 2003; 425, 191–196.

Haber CA, Lam TK, Yu Z, et al. N-Acetylcysteine and taurine prevent hyperglycemia-induced insulin resistance in vivo: possible role of oxidative stress. Am J Physi Endocrinol Metab 2003;285:E744– 753.

Hou DX, Uto T, Tong X, et al. Involvement of reactive oxygen species-independent mitochondrial pathway in gossypolinduced apoptosis. Arch Biochem Biophys 2004; 428: 179–187

Hung KY, Liu SY, Kao SH, et al. N-acetylcysteine-mediated antioxidation prevents hyperglycemia-induced apoptosis and collagen synthesis in rat mesangial cells. Am J Nephrol. 2009;29(3):192-202.

Isomaa B, Almgren P, Tuomi T, Forsen B, Lahti K, Nissen M, et al. Cardiovascular morbidity and mortality associated with the metabolic syndrome. Diabetes Care 2001; 24:683– 9.

Jang M, Cai L, Udeani GO, et al. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 1997; 275: 218–20.

Jang M, Pezzuto JM. Cancer chemopreventive activity of resveratrol. Drugs Exp Clin Res 1999; 25:65–77.

Juan ME, Vinardell MP, Planas JM. The Daily Oral Administration of High Doses of trans-Resveratrol to Rats for 28 Days Is Not Harmful. J. Nutr. 2002; 132: 257–260.

Kreisberg JI, Venkatachalam M, Troyer D. Contractile properties of cultured glomerular mesangial cells. Am J Physiol 1985; 249(4 Part 2):F457–F463.

Kulkarni AB, Huh CG., Becker D, et al. Transforming growth factor-b1 null mutation in mice causes excessive inflammatory response and early death.
Proc. Natl. Acad. Sci. 1993; 90: 770–774..

Kanakiriya SK, Croatt AJ, Haggard JJ ,et al. Heme: a novel inducer of MCP-1 through HO-dependent and HO-independent mechanisms. Am J Physiol Renal Physiol 2003; 284: F546–F554.
Kang BP, Frencher S, Reddy V, et al.: High glucose promotes mesangial cell apoptosis by oxidant dependent mechanism. Am J Physiol Renal Physiol 2003; 284:F455–F466.

Kang BP, Urbonas A, Baddoo A, et al.: IGF-1 inhibits the mitochondrial apoptosis program in mesangial cells exposed to high glucose. Am J Physiol Renal Physiol 2003;285: F1013–F1024.

Ko CH, Shen SC, Hsu CS, et al.: Mitochondrial- dependent, reactive oxygen species-independent apoptosis by myricetin: roles of protein kinase C, cytochrome c, and caspase cascade. Biochem Pharmacol 2005; 69: 913–927

Khera T, Martin J, Riley S, et al. Glucose enhances mesangial cell apoptosis. Lab Invest 2006; 86: 566–577.

Kirkby KA, Adin CA. Products of heme oxygenase and their potential therapeutic applications. Am J Physiol Renal Physiol 2006; 290: F563–F571.

Kim OS, Kim YS, Jang DS, et al..Cytoprotection against hydrogen peroxide-induced cell death in cultured mouse mesangial cells by erigeroflavanone, a novel compound from the flowers of Erigeron annuus. Chem Biol Interact. 2009; 180:414-20.

Lemos FB, Ijzermans JN, Zondervan PE, et al. Differential expression of heme oxygenase-1 and vascular endothelial growth factor in cadaveric and living donor kidneys after ischemia–reperfusion. J Am Soc Nephrol 2003; 14: 3278–3287.

Lin CL, Wang JY, Huang YT, et al. Wnt/β-Catenin Signaling Modulates Survival of High Glucose–Stressed Mesangial Cells. J Am Soc Nephrol. 2006 ;17:2812-20

Maines MD, Veltman JC. Phenylhydrazine-mediated induction of haem oxygenase activity in rat liver and kidney and development of hyperbilirubinaemia. Inhibition by zinc-protoporphyrin. Biochem J 1984; 217: 409–417

Maines MD, Mayer RD, Ewing JF, et al. Induction of kidney heme oxygenase-1 (HSP32) mRNA and protein by ischemia/reperfusion: possible role of heme as both promotor of tissue damage and regulator of HSP32. J Pharmacol Exp Ther 1993; 264: 457–462

McLennan SV, Fisher EJ, Yue DK, et al. High glucose concentration causes a decrease in mesangium degradation. Diabetes 1994;43:1041–1046.

Mishra R, Emancipator SN, Kern T, et al. High glucose evokes an intrinsic proapoptotic signaling pathway in mesangial cells. Kidney Int 2005; 67:82–93

Mishra R, Emancipator SN, Kern T, et al. High glucose evokes an intrinsic proapoptotic signaling pathway in mesangial cells. Kidney Int 2005; 67:82–93.

Nishikawa T, Edelstein D, Du XL, et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 2000; 404: 787–790.

Morales AI, Rodrı´guez-Barbero A, Vicente-Sa´nchez C, et al. Resveratrol inhibits gentamicin-induced mesangial cell contraction. Life Sciences 2006;78: 2373 – 2377

Nicholson DW, Thornberry NA: Apoptosis. Life and death decisions. Science 2003; 299: 214–215

Nath KA. Heme oxygenase-1: a provenance for cytoprotective pathways in the kidney and other tissues. Kidney Int 2006; 70: 432–443

Odetti P, Pesce C, Traverso N, et al. Comparative trial of N-acetyl-cysteine, taurine, and oxerutin on skin and kidney damage in long-term experimental diabetes.
Diabetes 2003; 52:499–505.

Pugliese G, Pricci F, Mene P .High glucose level unmasks a genetic predisposition to enhanced extracellular matrix production in mesangial cells from the Milan normotensive strain. J Am Soc Nephrol 1997; l 8:406–414

Powell LA, Nally SM, McMaster D, etal. Restoration of glutathione levels in vascular smooth muscle cells exposed to high glucose conditions. Free Radic Biol Med 2001;
31:1149– 55.

Pesce C, Menini S, Pricci F, et al. Glomerular cell replication and cell loss through apoptosis in experimental diabetes mellitus. Nephron 2002; 90:484–488

Pedraza-Chaverri J, Murali NS, Croatt AJ,et al. Proteinuria as a determinant of renal expression of heme oxygenase-1: studies in models of glomerular and tubular proteinuria in the rat. Am J Physiol Renal Physiol 2006; 290: F196–F204.

Steffes MW, Osterby R, Chavers B, et al. Mesangial expansion as a central mechanism for loss of kidney function in diabetic patients. Diabetes 1989; 38: 1077–1081.

Sharmak K, Ziyadefh F. The emerging role of transforming growth factor-β in kidney diseases. Am J Physiol 1994; 35: F829–F842

Sharma K, Ziyadeh FN. Hyperglycaemia and diabetic kidney disease. Diabetes 1995;44:1139–1146.

Sharma K, Jin Y, Guo J ,et al. Neutralization of TGF-β by anti-TGF-β antibody attenuates kidney hypertrophy and the enhanced extracellular matrix gene expression in STZ-induced diabetic mice. Diabetes 1996; 45: 522–530

Slater I, Odum J, Ashby J. Resveratrol and red wine consumption. Hum Exp Toxicol 1999;18:625–6.

Shimizu H, Takahashi T, Suzuki T, et al. Protective effect of heme oxygenase induction in ischemic acute renal failure. Crit Care Med 2000; 28: 809–817.

Shiraishi F, Curtis LM, Truong L, et al. Heme oxygenase-1 gene ablation or expression modulates cisplatin-induced renal tubular apoptosis. Am J Physiol Renal Physiol 2000; 278: F726–F736

Sono M, Chen S, Hong SW, et al. Smad pathway is activated in the diabetic mouse kidney and Smad3 mediates TGF-β-induced fibronectin in mesangial cells. Biochem Biophys Res Commun 2002; 296: 1356–1365

Sinclair, D. A. Toward a unified theory of caloric restriction and longevity regulation. Mech. Ageing Dev. 2005; 126: 987–1002

Suenaga F, Hatsushika K, Takano S, et al: A possible link between resveratrol and TGF-β: Resveratrol induction of TGF-βexpression and signaling. FEBS Letters 2008; 582: 586-590

Tetsuka T, Daphna-Iken D, Srivastava SK, et al. Regulation of heme oxygenase mRNA in mesangial cells: prostaglandin E2 negatively modulates interleukin-1-induced heme oxygenase-1 mRNA. Biochem Biophys Res Commun 1995; 212: 617–623.

The diabetes control and complications trial/epidemiology of diabetes intervention and complications research group. Retinopathy and nephropathy in patients with type 1 diabetes four years after a trial of intensive therapy. N Engl J Med 2000;342:381–389.

USRDS 2009 Annual Data Report. www.usrds.org

Venkatesan B, Ghosh-Choudhury N, Das F, et al. Resveratrol inhibits PDGF receptor mitogenic signaling in mesangial cells: role of PTP1B . FASEB J. 2008 ;22(10):3469-82.

Valenzano DR. et al. Resveratrol prolongs lifespan and retards the onset of age related markers in a short-lived vertebrate. Curr. Biol. 2006;16, 296–300.

Wolf G, Sharma K, Chen Y, et al. High glucose-induced proliferation in mesangial cells is reversed by autocrine TGF-b. Kidney Int 1992; 42:647–656.

White KE, Bilous RW. Type 2 diabetic patients with nephropathy show structural-functional relationships that are similar to type 1 disease. J Am Soc Nephrol
2000;11:1667–1673.

White KE, Bilous RW, Marshall SM, et al. Podocyte number in normotensive type 1 diabetic patients with albuminuria. Diabetes 2002; 51:3083–3089.

Yue KK, Chung WS, Leung AW, etal. Redox changes precede the occurrence of oxidative stress in eyes and aorta, but not in kidneys of diabetic rats. Life Sci 2003; 73:2557– 70.

Ziyadeh FN. Mediators of hyperglycemia and the pathogenesis of matrix accumulation in diabetic renal disease. Miner Electrolyte Metab 1995; 21: 292–302.

Zou JG, Huang YZ, Chen Q,et al. Resveratrol inhibits copper ion-induced and azo compound-initiated oxidative modification of human low density lipoprotein. Biochem Mol Biol Int 1999;47:1089–96.

------------------------------------------------------------------------ 第 8 筆 ---------------------------------------------------------------------
系統識別號 U0007-1805200916334300
論文名稱(中文) Dipyridamole對抗腎臟發炎與缺氧作用之研究
論文名稱(英文) The Effects of Dipyridamole on Renal Inflammation and Hypoxia
校院名稱 臺北醫學大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Graduate Institute of Clinical Medicine
學年度 97
學期 2
出版年 98
研究生(中文) 陳作孝
學號 D102091012
學位類別 博士
語文別 中文
口試日期 2009-05-07
論文頁數 99頁
口試委員 指導教授-陳保羅
共同指導教授-李宏謨
委員-樓迎統
委員-陳振文
委員-林裕峯
委員-陳漢湘
關鍵字(中) Dipyridamole, 血红
素氧合酶
1, 分裂素蛋白激酶
磷酸酶
1, 骨橋素
關鍵字(英) Dipyridamole, Heme oxygenase-1, Mitogen-activated kinase phosphatase-1, Osteopontin
學科別分類
中文摘要 研究背景. 慢性腎臟病的病理特徵包括腎絲球硬化、腎間質白血球浸潤、腎小管間質纖維化、腎絲球及腎小管周邊微血管喪失。任何形式的腎臟損傷會造成發炎物質的生成,微血管喪失引起組織的缺氧缺血,產生反應性氧族(ROS),進一步強化發炎反應、組織的修補和纖維化。腎臟慢性的缺氧會活化腎素-血管收縮素系統,局部的血管收縮素II增加會引起氧化壓力。同時,腎臟組織的缺氧缺血反應會增加血红素氧合酶1的表現,提供腎臟損傷的保護機制。Dipyridamole是一個核苷運送的抑制劑,也是一個非選擇性磷酸二酯酶(phosphodiesterase)的抑制劑。Dipyridamole應用在腎臟病治療可改善蛋白尿,然而,作用的機轉並不清楚。本研究將探討dipyridamole 抗發炎及抗氧化的作用,並研究活化分裂素蛋白激酶磷酸酶1 (MKP-1)及血红素氧合酶1 (HO-1)扮演的角色。
研究方法. 白細胞介素6 (interleukin 6, 簡稱IL-6)、單核球趨化蛋白-1 (monocyte chemoattractant protein, 簡稱MCP-1)及骨橋素(osteopontin, 簡稱OPN) 以ELISA方法測量。ROS的產量以(DCF)螢光強度測量。研究PI-3K-PKB/Akt,分裂素蛋白激酶(MAPK) 和NF-kB 訊息傳遞路徑。利用分裂素蛋白激酶磷酸酶1和血红素氧合酶1的短鏈干擾核糖核酸(siRNA)轉殖的細胞,抑制MKP-1及 HO-1基因表現,來研究dipyridamole的分子作用機制。
結果. 在大鼠腎膈細胞(RMCs),加入100nM的血管收縮素II會增加ROS的產生,PI-3K的抑制劑 Ly294001及wortmannin會阻斷血管收縮素II所誘導HIF-1α堆積,顯示血管收縮素II是透過ROS依賴的PI-3K-PKB /Akt 路徑,誘導HIF-1α堆積。在大鼠巨噬細胞(RAW 264.7 cells),dipyridamole可以活化MKP-1,以MKP-1 siRNA轉殖技術抑制MKP-1基因表現或加入MKP-1的抑制劑 triptolide,可以抑制脂多醣體(lipopolysaccharide,LPS)在巨噬細胞所誘導環氧化酵素2 (COX-2)的表現。在大鼠腎膈細胞,我們同樣發現dipyridamole可以抑制脂多醣體所誘導IL-6和MCP-1分泌,ROS的產量有明顯的減少且和dipyridamole劑量相關。另外,dipyridamole會抑制脂多醣體引起的NF-kB和脫氧核醣核酸結合能力及IkB的磷酸化。以MKP-1 siRNA轉殖的大鼠腎膈細胞則會降低dipyridamole對脂多醣體所誘導IL-6表現的抑制作用。ERK 1/2 及p38MAPK 路徑也參與了脂多醣體所引發的MCP-1和COX-2的表現,可以受到dipyridamole及抗氧化劑 l-NAC所抑制。然而,以SnPP (HO-1抑制劑)前處理RMC細胞,可逆轉dipyridamole對ROS及發炎反應的抑制作用。在大鼠腎小管上皮細胞(NRK-52E),加入化學缺氧劑氯化鈷(CoCl2),會增加OPN蛋白質表現增加。Dipyridamole可以誘導HO-1的表現增加與抑制CoCl2所誘導OPN的分泌,以SnPP或血色素(一氧化碳去除劑),可逆轉dipyridamole對OPN的抑制作用,顯示HO-1與dipyridamole抑制CoCl2誘導OPN的表現有關。以HO-1 siRNA轉殖的NRK-52E細胞會降低dipyridamole所誘導MKP-1的磷酸化及活化,而以MKP-1 siRNA轉殖的NRK-52E細胞會則能逆轉dipyridamole對OPN的抑制作用。
結論. 我們的實驗結果顯示,dipyridamole會先藉由活化MKP-1的方式使得p38 MAPK去磷酸化而失去功能。此外,血管收縮素II透過ROS依賴的路徑來誘導HIF-1α堆積。在大鼠腎膈細胞,dipyridamole藉由HO-1降低ROS ,抑制脂多醣體所誘導COX-2表現及MCP-1分泌。在NRK-52E細胞,dipyridamole會經由誘導HO-1及MKP-1的活化,降低CoCl2所誘導OPN的分泌。總而言之, dipyridamole可能透過抗發炎及抗氧化的作用,提供在慢性腎臟病治療的好處。
英文摘要 Background. Chronic kidney disease is characterized by glomerulosclerosis, interstitial leukocyte infiltration, tubulointerstitial fibrosis, and loss of glomerular and peritubular capillaries. Renal injury of any kind generates mediators of inflammation. Hypoxia/ischemia secondary to loss of capillaries also contributes to reactive oxygen species (ROS) generation and may further enhance the inflammatory process, tissue remodeling and fibrosis. Chronic hypoxia can activate the renin-angiotensin system (RAS) and local angiotensin II induces oxidative stress. In the meantime, up-regulation of heme ogygenase-1 (HO-1) provides protection against renal injury that follows hypoxia/ischemia. Dipyridamole is a nucleoside transport inhibitor and a non-selective phosphodiesterase inhibitor, which has been shown to improve proteinuria. However, the mechanisms by which dipyridamole exerts its beneficial effects on renal disease are not completely understood. In the present study, we investigated the roles of mitogen-activated kinase phosphatase-1 (MKP-1) and HO-1 in dipyridamole's anti-inflammatory and anti-oxidative effects.
Methods. IL-6, MCP-1 and OPN secretions were measured by ELISA kits. ROS generation was measured using the fluorescent probe 2',7'- dichlorofluorescein (DCF). PI-3K-PKB/Akt, MAPK and NF-kB signal pathways were studied. HO-1 and MKP-1 siRNA were used for gene knockdown to investigate the molecular mechanisms of dipyridamole.
Results. Treatment of rat mesangial cells (RMCs) with Ang II (100 nM) increased production of ROS. Ang II-stimulated HIF-1alpha accumulation was blocked by the phosphatidylinositol 3-kinase (PI-3K) inhibitors, Ly 294001, and wortmannin, suggesting that Ang II may stimulate a ROS-dependent activation of the PI-3K-PKB/Akt pathway, which leads to HIF-1alpha accumulation. In RAW 264.7 cells, dipyridamole stimulated transient activation of MKP-1, a potent inhibitor of p38 MAPK function. Knockdown of MKP-1 by transfecting MKP-1 siRNA or inhibition of MKP-1 by the specific inhibitor, triptolide, significantly reduced the inhibitory effects of dipyridamole on COX-2 expression induced by LPS. We also showed that dipyridamole inhibited LPS-induced IL-6 and MCP-1 secretion in RMCs. Pretreated with dipyridamole showed significantly inhibition of ROS generation in a dose-dependent manner. In addition, dipyridamole inhibited the LPS-stimulated NF-kB DNA binding activity and IkB phosphorylation. The dipyridamole inhibitory effect on LPS-induced IL-6 secretion was reduced in MKP-1 siRNA knockdown cells. ERK1/2 and p38 MAPK signaling pathways were demonstrated to be involved in the LPS-induced MCP-1 secretion and COX-2 expression, and were inhibited by dipyridamole and l-NAC treatment. However, pretreatment of RMCs with tin protoporphyrin (Snpp; an HO-1 inhibitor) reversed the inhibitory effect of dipyridamole on ROS and inflammatory responses. Incubation of rat renal tubular NRK52E cells with cobalt chloride (CoCl2) increased OPN production. Dipyridamole could induce HO-1 expression and inhibited CoCl2-induced OPN secretion. Pretreatment of cells with Snpp, or hemoglobin (a CO-scavenging agent), reversed the inhibition of OPN expression by dipyridamole. Transfection of HO-1 siRNA reduced dipyridamole-stimulated MKP-1 phosphorylation. Knockdown of MKP-1 reversed the inhibition of OPN expression by dipyridamole.
Conclusions. Our results demonstrate that dipyridamole may exert its anti-inflammatory effect via activation of MKP-1, which dephosphorylates and inactivates p38 MAPK. In addition, Ang II increases ROS-dependent HIF-1 alpha accumulation. Dipyridamole inhibits the expression of COX-2 and secreted MCP-1 in LPS-treated RMCs via HO-1-mediated ROS reduction. In NRK-52E cells, dipyridamole may suppress CoCl2-induced OPN secretion via induction of HO-1 and activation of MKP-1. Taken together, these data suggest that dipyridamole may contribute to its beneficial effects on chronic kidney disease through its anti-inflammatory and anti-oxidative effects.
論文目次 縮寫表 (Abbreviations) ------------------------------- iv
圖目錄 (Figures Contents) ---------------------------- vi
中文摘要 (Abstract in Chinese) ----------------------- ix
英文摘要 (Abstract in English) ---------------------- xii
第一章 緒論 (Introduction)
第一節 慢性腎臟病的病理變化與臨床重要性--------------- 1
第二節 Dipyridamole的藥理作用與臨床應用--------------- 4
第三節 分裂素活化蛋白激酶磷酸酶1 (MKP-1) ------------ 6
第四節 低氧症可誘導因素1 (HIF-1) -------------------- 7
第五節 血红素氧合酶1 (HO-1) -------------------------- 8
第六節 骨橋素 ---------------------------------------- 9
第七節 研究目的 ------------------------------------- 10
第二章 研究方法與材料 (Materials and Methods) -------- 11
第三章 研究結果 (Results)
第一節 血管收縮素II在大鼠腎膈細胞誘導HIF-1α堆積 ------ 19
第二節 Dipyridamole經由活化MKP-1抑制脂多醣體在巨噬細胞 所誘導環氧化酵素2的表現 ------------------------------ 21
第三節 Dipyridamole經由活化MKP-1抑制脂多醣在大鼠腎膈細胞 所誘導白細胞介素6的表現 ------------------------------ 25
第四節 Dipyridamole經由HO-1降低反應性氧族抑制脂多醣體在 大鼠腎膈細胞所誘導環氧化酵素2及單核細胞趨化素1的表現-- 27
第五節 Dipyridamole經由HO-1抑制大鼠腎小管上皮細胞骨橋素 的表現 ----------------------------------------------- 31
第四章 討論 (Discussion)
第一節 血管收縮素II在大鼠腎膈細胞誘導HIF-1α堆積 ------ 35
第二節 Dipyridamole經由活化MKP-1抑制脂多醣體 在巨噬細胞所誘導環氧化酵素2的表現 -------------------- 38
第三節 Dipyridamole經由活化MKP-1抑制脂多醣在大鼠腎膈細胞 所誘導白細胞介素6的表現 ------------------------------ 43
第四節 Dipyridamole經由HO-1降低反應性氧族抑制脂多醣體在 大鼠腎膈細胞所誘導環氧化酵素2及單核細胞趨化素1的表現 - 46
第五節 Dipyridamole經由HO-1抑制大鼠腎小管上皮細胞骨橋素 的表現 ----------------------------------------------- 50
第五章 結論與展望 (Conclusion and Perspective) ------- 55 參考文獻 (References) ------------------------------- 57
圖表 (Tables and Figures) ---------------------------- 74
參考文獻 Abbot, F., Ryan, J.J., Ceska, M., Matsushima, K., Sarra, C.E., Rees, A.J., 1991. Interleukin-1 beta stimulates human mesangial cells to synthesize and release interleukin-6 and -8. Kidney Int 40:597-605
Aiello, L.P., 2000. Role of vascular endothelial growth factor in diabetic vascular complications. Kidney Int 77: S113–S119
Alvarez-Maqueda, M., El Bekay, R., Alba, G., Monteseirin, J., Chacon, P., Vega, A., Martin-Nieto, J., Bedoya, F.J., Pintado, E., Sobrino, F., 2004. 15-deoxy-delta 12,14-prostaglandin J2 induces heme oxygenase-1 gene expression in a reactive oxygen species-dependent manner in human lymphocytes. J Biol Chem 279: 21929-21937
Ardyanto, T.D., Osaki, M., Tokuyasu, N., Nagahama, Y., Ito, H., 2006. CoCl2-induced HIF-1alpha expression correlates with proliferation and apoptosis in MKN-1 cells: a possible role for the PI3K/Akt pathway. Int J Oncol 29: 549-555
Ashkar, S., Weber, G.F., Panoutsakopoulou, V., Sanchirico, M.E., Jansson, M., Zawaideh, S., Rittling, S.R., Denhardt, D.T., Glimcher, M.J., Cantor, H., 2000. Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity. Science 287: 860-864
Awazu, M., Omori, S., Hida, M., 2002. MAP kinase in renal development. Nephrol Dial Transplant 17(Suppl 9):5-7
Baldwin, A.S., 1996. The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu Rev Immunol 14:649-656
Basile, D.P., Fredrich, K., Alausa, M., Vio, C.P., Liang, M., Rieder, M.R., Greene, A.S., Cowley, A.W., Jr., 2005. Identification of persistently altered gene expression in the kidney after functional recovery from ischemic acute renal failure. Am J Physiol Renal Physiol 288: F953-963
Beavo, J.A., 1995. Cyclic nucleotide phosphodiesterases: functional implications of multiple isoforms. Physiol Rev 75:725-732
Bender, A.T., Beavo, J.A., 2006. Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol Rev 58: 488-520
Bielekova, B., Lincoln, A., McFarland, H., Martin, R., 2000. Therapeutic potential of phosphodiesterase-4 and -3 inhibitors in Th1-mediated autoimmune diseases. J Immunol 164:1117-1130
Bowie, A., O'Neill, L.A., 2000. Oxidative stress and nuclear factor-kappaB activation: a reassessment of the evidence in the light of recent discoveries. Biochem Pharmacol 59:13-20
Brown, K., Gerstberger, S., Carlson, L., Franzoso, G., Siebenlist, U., 1995. Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation. Science 267:1485-1492
Burnouf, C., Pruniaux, M.P., 2002. Recent advances in PDE4 inhibitors as immunoregulators and anti-inflammatory drugs. Curr Pharm Des 8:1255-1265
Camara, S., Cruz, J.P., Frutos, M.A., Sanchez, P., Lopez de Novales, E., Sanchez, E., Sanchez de la Cuesta, F., 1991. Effects of dipyridamole on the short-term evolution of glomerulonephritis. Nephron 58: 13-16
Camhi, S.L., Alam, J., Otterbein, L., Sylvester, S.L., Choi, A.M., 1995. Induction of heme oxygenase-1 gene expression by lipopolysaccharide is mediated by AP-1 activation. Am J Respir Cell Mol Biol 13: 387-398
Chandel, N.S., McClintock, D.S., Feliciano, C.E., Wood, T.M., Melendez, J.A., Rodriguez, A.M., Schumacker, P.T., 2000. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing. J Biol Chem 275: 25130-25138
Chandel, N.S., 2000. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1α during hypoxia. J Biol Chem 275: 25130–25138
Chakrabarti, S., Vitseva, O., Iyu, D., Varghese, S., Freedman, J.E., 2005. The effect of dipyridamole on vascular cell-derived reactive oxygen species. J Pharmacol Exp Ther 315: 494–500
Chen, T.H., Kao, Y.C., Chen, B.C., Chen, C.H., Chan, P., Lee, H.M., 2006. Dipyridamole activation of mitogen-activated protein kinase phosphatase-1 mediates inhibition of lipopolysaccharide-induced cyclooxygenase-2 expression in RAW 264.7 cells. Eur J Pharmacol 541: 138-146
Chen, Z., Hagler, J., Palombella, V.J., 1995. Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway. Genes Dev 9:1586-1595
Chen, P., Li, J., Barnes, J., Kokkonen, G.C., Lee, J.C., Liu, Y., 2002. Restraint of proinflammatory cytokine biosynthesis by mitogen- activated protein kinase phosphatase-1 in lipopolysaccharide- stimulated macrophages. J Immunol 169:6408-6418
Cheng, J., Grande, J.P., 2007. Cyclic nucleotide phosphodiesterase (PDE) inhibitors: novel therapeutic agents for progressive renal disease. Exp Biol Med (Maywood) 232: 38-51
Choi, A.M., Alam, J., 1996. Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury. Am J Respir Cell Mol Biol 15: 9-19
Choi, J.S., Kim, H.Y., Cha, J.H., Choi, J.Y., Lee, M.Y., 2007. Transient microglial and prolonged astroglial upregulation of osteopontin following transient forebrain ischemia in rats. Brain Res 1151: 195-202
Coleman, D., Ruef, C., 1992. Interleukin-6: An autocrine regulator of mesangial cell growth. Kidney Int 41: 604–611
Cuturi, M.C., Christoph, F., Woo, J., Iyer, S., Brouard, S., Heslan, J.M., Pignon, P., Soulillou, J.P., Buelow, R. 1999. RDP1258, a new rationally designed immunosuppressive peptide, prolongs allograft survival in rats: analysis of its mechanism of action. Mol Med 5: 820-832
Dastidar, S.G., Rajagopal, D., Ray, A., 2007. Therapeutic benefit of PDE4 inhibitors in inflammatory diseases. Curr Opin Investig Drugs 8: 364-372
Denhardt, D.T., Guo, X., 1993. Osteopontin: a protein with diverse functions. FASEB J 7: 1475-1482
Dubey, R.K., Gillespie, D.G., Jackson, E.K., 1999. Adenosine inhibits collagen and total protein synthesis in vascular smooth muscle cells. Hypertension 33: 190-194
Eddy, A.A., 1994. Experimental insights into the tubulointerstitial disease accompanying primary glomerular lesions. J Am Soc Nephrol 5: 1273–1287
Eddy, A.A., 2005. Progression in Chronic Kidney Disease. Advances in Chronic Kidney Disease 12:353-365
Enomoto, N., 2002. Hypoxic induction of hypoxia-inducible factor-1α and oxygen-regulated gene expression in mitochondrial DNA-depleted HeLa cells. Biochem Biophys Res Commun 297: 346–352
Endo, Y., Kanbayashi, H., Hara, M., 1993. Experimental immunoglobulin A nephropathy induced by gram-negative bacteria. Nephron 65: 196–205
Gaedeke, J., Noble, N.A., Border, W.A., 2005. Curcumin blocks fibrosis in anti-Thy 1 glomerulonephritis through up-regulation of heme oxygenase 1. Kidney Int 68: 2042-2049
Gaitanaki, C., Kalpachidou, T., Aggeli, I.K., Papazafiri, P., Beis, I., 2007. CoCl2 induces protective events via the p38-MAPK signalling pathway and ANP in the perfused amphibian heart. J Exp Biol 210: 2267-2277
Giachelli, C.M., Pichler, R., Lombardi, D., Denhardt, D.T., Alpers, C.E., Schwartz, S.M., Johnson, R.J., 1994. Osteopontin expression in angiotensin II-induced tubulointerstitial nephritis. Kidney Int 45: 515-524
Gloire, G., Legrand-Poels, S., Piette, J., 2006. NF-kappaB activation by reactive oxygen species: fifteen years later. Biochem Pharmacol 72: 1493-1505
Gomez, G.C., Lopez, M.J., Gonzalez, E., Egido, J., 1994. Soluble IgA and IgG aggregates are catabolized by cultured rat mesangial and induce production of TNF- and IL-6 and proliferation. J Immunol 154: 5247–5256
Graf, K., Stawowy, P., 2004. Osteopontin: a protective mediator of cardiac fibrosis? Hypertension 44: 809-810
Granholm, N.A., Cavallo, T., 1994. Long-lasting effects of bacterial lipopolysaccharide promote progression of lupus nephritis in NZB/W mice. Lupus 3: 507–514
Hammond, J.R., Williams, E.F., Clanachan, A.S., 1985. Affinity of calcium channel inhibitors, benzodiazepines, and other vasoactive compounds for the nucleoside transport system. Can J Physiol Pharmacol 63: 1302-1307
Hammond, J.R., Stolk, M., Archer, R.G., McConnell, K., 2004. Pharmacological analysis and molecular cloning of the canine equilibrative nucleoside transporter 1. Eur J Pharmacol 491: 9-19
Harmankaya, O., Basturk, T., Ozturk, Y., Karabiber, N., Obek, A., 2001. Effect of acetylsalicylic acid and dipyridamole in primary membranoproliferative glomerulonephritis type I. Int Urol Nephrol 33: 583-587
Haas, C.S., Schocklmann, H.O., Lang, S., Kralewski, M., Sterzel, R.M., 1999. Regulatory mechanism in glomerular mesangial cell proliferation. J Nephrol 12: 405-15
Haas, C., Car, B., Ryffel, B.E., Hir, M., 1996. Lipopolysaccharide-induced glomerulonephritis develops in the absence of interferon-gamma signaling. Exp Nephrol 64: 222–230
Maxwell. P., 2003. HIF-1: An ogygen response system with specialy relevance to th kidney. J Am Soc Nephrol 14:2712-2722

Hewitson, T.D., Tait, M.G., Kelynack, K.J., Martic, M., Becker, G.J., 2002. Dipyridamole inhibits in vitro renal fibroblast proliferation and collagen synthesis. J Lab Clin Med 140: 199-208
Higgins, D.F., Biju. M.P., Akai, Y., Wutz, A., Johnson, R.S., Haase, V.H., 2004. Hypoxic induction of Ctgf is directly mediated by Hif-1. Am J Physiol Renal Physio 287(6):F1223-32
Hung, K.Y., Chen, C.T., Huang, J.W., Lee, P.H., Tsai, T.J., Hsieh, B.S., 2001. Dipyridamole inhibits TGF-beta-induced collagen gene expression in human peritoneal mesothelial cells. Kidney Int 60: 1249-1257
Hung, K.Y., Chen, C.T., Yen, C.J., Lee, P.H., Tsai, T.J., Hsieh, B.S., 2001. Dipyridamole inhibits PDGF-stimulated human peritoneal mesothelial cell proliferation. Kidney Int 60: 872-881
Hung, K.Y., Shyu, R.S., Fang, C.C., Tsai, C.C., Lee, P.H., Tsai, T.J., Hsieh, B.S., 2001c. Dipyridamole inhibits human peritoneal mesothelial cell proliferation in vitro and attenuates rat peritoneal fibrosis in vivo. Kidney Int 59: 2316-2324
Huang, L.E., 2002. Leu-574 of HIF-1α is essential for the von Hippel-Lindau (VHL)-mediated degradation pathway. J Biol Chem 277: 41750–41755
Iguchi, S., Nishi, S., Ikegame, M., Hoshi, K., Yoshizawa, T., Kawashima, H., Arakawa, M., Ozawa, H., Gejyo, F., 2004. Expression of osteopontin in cisplatin-induced tubular injury. Nephron Exp Nephrol 97: e96-105


Ikeda, M., Ikeda, U., Ohkawa, F., Shimada, K., Kano, S., 1994. Nitric oxide synthesis in rat mesangial cells induced by cytokines. Cytokine 6: 602-607
Iuliano, L., Pedersen, J.Z., Rotilio, G., Ferro, D., Violi, F., 1995. A potent chain-breaking antioxidant activity of the cardiovascular drug dipyridamole. Free Radic Biol Med 18: 239–247
Iuliano, L., Colavita, A.R., Camastra, C., Bello, V., Quintarelli, C., Alessandroni, M., Piovella, F., Violi, F., 1996. Protection of low density lipoprotein oxidation at chemical and cellular level by the antioxidant drug dipyridamole. Br J Pharmacol 119: 1438–1446
Jin, H.O., An, S., Lee, H.C., Woo, S.H., Seo, S.K., Choe, T.B., Yoo, D.H., Lee, S.B., Um, H.D., Lee, S.J., Park, M.J., Kim, J.I., Hong, S.I., Rhee, C.H., Park, I.C., 2007. Hypoxic condition- and high cell density-induced expression of Redd1 is regulated by activation of hypoxia-inducible factor-1alpha and Sp1 through the phosphatidylinositol 3-kinase/Akt signaling pathway. Cell Signal 19: 1393-1403
Karkar, A.M., Rees, A.J., 1997. Influence of endotoxin contamination on anti-GBM antibody induced glomerular injury in rats. Kidney Int 52: 1579–1583
Khanna, A., Plummer, M., Bromberek, C., Bresnahan, B., Hariharan, S., 2002. Expression of TGF-beta and fibrogenic genes in transplant recipients with tacrolimus and cyclosporine nephrotoxicity. Kidney Int 62: 2257-2263
Kitamura, Y., Matsuoka, Y., Nomura, Y., Taniguchi, T., 1998. Induction of inducible nitric oxide synthase and heme oxygenase-1 in rat glial cells. Life Sci 62: 1717-1721
Kim, S., 2000. Molecular and cellular mechanisms of angiotensin II-mediated cardiovascular and renal diseases. Pharmacol. Rev 52: 11–34
Komers, R., Epstein, M., 2002. Cyclooxygenase-2 expression and function in renal pathophysiology. J Hypertens 20: S11-15
Kossmehl, P., Schonberger, J., Shakibaei, M., Faramarzi, S., Kurth, E., Habighorst, B., von Bauer, R., Wehland, M., Kreutz, R., Infanger, M., Schulze-Tanzil, G., Paul, M., Grimm, D., 2005. Increase of fibronectin and osteopontin in porcine hearts following ischemia and reperfusion. J Mol Med 83: 626-637
Kramer, A.B., Ricardo, S.D., Kelly, D.J., Waanders, F., van Goor, H., Navis, G., 2005. Modulation of osteopontin in proteinuria-induced renal interstitial fibrosis. J Pathol 207: 483-492
Kusmic, C., Picano, E., Busceti, C.L., Petersen, C., Barsacchi, R., 2000. The antioxidant drug dipyridamole spares the vitamin E and thiols in red blood cells after oxidative stress. Cardiovasc Res 47: 510–514
Lan, H.Y., Yu, X.Q., Yang, N., Nikolic-Paterson, D.J., Mu, W., Pichler, R., Johnson, R.J., Atkins, R.C., 1998. De novo glomerular osteopontin expression in rat crescentic glomerulonephritis. Kidney Int 53: 136-145
Levin, A., 1999.Management of membranoproliferative glomerulonephritis: evidence-based recommendations. Kidney Int 70: S41-46
Leonard, M., Ryan, M.P., Watson, A.J., Schramek, H., Healy, E., 1999. Role of MAP kinase pathways in mediating IL-6 production in human primary mesangial and proximal tubular cells. Kidney Int 56(4): 1366-1377
Li, X.Y., Zhang, C., Wang, S.F., Ji, Y.L., Wang, H., Zhao, L., Xu, D.X., 2008. Maternally administered lipopolysaccharide (LPS) upregulates the expression of heme oxygenase-1 in fetal liver: The role of reactive oxygen species. Toxicol Lett 176: 169-177
Li, C., Sun, B.K., Lim, S.W., Song, J.C., Kang, S.W., Kim, Y.S., Kang, D.H., Cha,J.H., Kim,J., Yang,C.W., 2005. Combined effects of losartan and pravastatin on interstitial inflammation and fibrosis in chronic cyclosporine-induced nephropathy. Transplantation 79: 1522-1529
Liaw, L., Lindner, V., Schwartz, S.M., Chambers, A.F., Giachelli, C.M., 1995. Osteopontin and beta 3 integrin are coordinately expressed in regenerating endothelium in vivo and stimulate Arg-Gly-Asp- dependent endothelial migration in vitro. Circ Res 77: 665-672
Loboda, A., Jazwa, A., Wegiel, B., Jozkowicz, A., Dulak, J., 2005. Heme oxygenase-1-dependent and -independent regulation of angiogenic genes expression: effect of cobalt protoporphyrin and cobalt chloride on VEGF and IL-8 synthesis in human microvascular endothelial cells. Cell Mol Biol 51: 347-355
Lorena, D., Darby, I.A., Gadeau, A.P., Leen, L.L., Rittling, S., Porto, L.C., Rosenbaum, J., Desmouliere, A., 2006. Osteopontin expression in normal and fibrotic liver. altered liver healing in osteopontin-deficient mice. J Hepatol 44: 383-390
Lu, N., Zhou, H., Lin, Y.H., Chen, Z.Q., Pan, Y., Li, X.J., 2007. Oxidative stress mediates CoCl(2)-induced prostate tumour cell adhesion: role of protein kinase C and p38 MAPK. Basic Clin Pharmacol Toxicol 101: 41-46
Marieke, E.A., Fokko, W., Willeke, M.S., Leendert, E., Mohamed, R.D., 1993. Soluble aggregates of IgG and immune complexes enhance IL-6 production by renal mesangial cells. Kidney Int 43: 544–553
Marina, L., Sonya, M., Christine, B., Jeremy, S., Andrew, R.C., 2002. Dexamethasone causes sustained expression of mitogen-activated protein kinase (MAPK) phosphatase 1 and phosphatase-mediated inhibition of MAPK p38. Mol Cell Biol 22:7802-7811
Maines, M.D., Mayer, R.D., Ewing, J.F., McCoubrey, W.K., 1993. Induction of kidney heme oxygenase-1 (HSP32) mRNA and protein by ischemia/reperfusion: possible role of heme as both promotor of tissue damage and regulator of HSP32. J Pharmacol Exp Ther 264: 457-462
Malyankar, U.M., Almeida, M., Johnson, R.J., Pichler, R.H., Giachelli, C.M., 1997. Osteopontin regulation in cultured rat renal epithelial cells. Kidney Int 51: 1766-1773
Mark, A., Hock, T., Kapturczak, M.H., Agarwal, A., Hill-Kapturczak, N., 2005. Induction of heme oxygenase-1 modulates the profibrotic effects of transforming growth factor-beta in human renal tubular epithelial cells. Cell Mol Biol 51: 357-362
Montellano, P.R., 2000. The mechanism of heme oxygenase. Curr Opin Chem Biol 4: 221-227

Morse, D., Choi, A.M., 2002. Heme oxygenase-1: the "emerging molecule" has arrived. Am J Respir Cell Mol Biol 27: 8-16
Nath, K.A., Grande, J.P, Haggard, J.J., Croatt, A.J., Katusic, Z.S., Solovey, A., Hebbel, R.P., 2001. Oxidative stress and induction of heme oxygenase-1 in the kidney in sickle cell disease. Am J Pathol 158: 893-903
Nath, K.A., Haggard, J.J., Croatt, A.J., Grande, J.P., Poss, K.D., Alam, J., 2000. The indispensability of heme oxygenase-1 in protecting against acute heme protein-induced toxicity in vivo. Am J Pathol 156: 1527-1535
Nath, K.A., Balla, G., Vercellotti, G.M., Balla, J., Jacob, H.S., Levitt, M.D., Rosenberg, M.E., 1992. Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat. J Clin Invest 90: 267-270
Nakamura, A., Suzuki, T., Kohsaka, T., 1995. Renal tubular function modulates urinary levels of Interleukin-6. Nephron 70: 416–420
Nolin, L., Courteau, M., 1999. Management of IgA nephropathy: evidence-based recommendations. Kidney Int 70: S56-62
Norman, J.T., Clark, I.M., Garcia, P.L., 2000. Hypoxia promotes fibrogenesis in human renal fibroblasts. Kidney Int 58: 2351-2366
Otterbein, L.E., Choi, A.M., 2000. Heme oxygenase: colors of defense against cellular stress. Am J Physiol Lung Cell Mol Physiol 279: L1029-1037
Pardo, A., Gibson, K., Cisneros, J., Richards, T.J., Yang, Y., Becerril, C., Yousem, S., Herrera, I., Ruiz, V., Selman, M., Kaminski, N., 2005. Up-regulation and profibrotic role of osteopontin in human idiopathic pulmonary fibrosis. Plos Med 2: e251
Papayianni, A., 1996. Cytokines, growth factors, and other inflammatory mediators in glomerulonephritis. Renal Failure 18:725-740
Page, E.L., 2002. Induction of hypoxia-inducible factor-1α by transcriptional and translational mechanisms. J Biol Chem 277: 48403–48409
Panzer, U., Thaiss, F., Zahner, G., Barth, P., Reszka, M., Reinking, R.R., Wolf, G., Helmchen, U., Stahl, R.A., 2001. Monocyte chemoattractant protein-1 and osteopontin differentially regulate monocytes recruitment in experimental glomerulonephritis. Kidney Int 59: 1762-1769
Pichler, R.H., Franceschini, N., Young, B.A., Hugo, C., Andoh, T.F., Burdmann, E.A., Shankland, S.J., Alpers, C.E., Bennett, W.M., Couser, W.G., et al., 1995. Pathogenesis of cyclosporine nephropathy: roles of angiotensin II and osteopontin. J Am Soc Nephrol 6: 1186-1196
Radeke, H.H., Resch, K., 1992. The inflammatory function of renal glomerular mesangial cells and their interaction with the cellular immune system. Clin Investig 70: 825-842
Rastaldi, M.P., Ferrario, F., Crippa, A., 2000. Glomerular monocyte-macrophage features in ANCA-positive renal vasculitis and cryoglobulinemic nephritis. J Am Soc Nephrol 11: 2036–2043
Risau, W., 1997. Mechanisms of angiogenesis. Nature 386: 671–674
Rovin, B.H., Dickerson, J.A., Tan, L.C., Hebert, C.A., 1995. Activation of nuclear factor-kappa B correlates with MCP-1 expression by human mesangial cells. Kidney Int 48: 1263–1271
Segerer, S., Kretzler, M., Strutz. F., 2007. Mechanisms of tissue injury and repair in renal diseases. In: Schrier R (ed). Diseases of the Kidney and Urinary Tract. Lippincott, Philadelphia
Shimizu, H., Takahashi, T., Suzuki, T., Yamasaki, A., Fujiwara, T., Odaka, Y., Hirakawa, M., Fujita, H., Akagi. R., 2000. Protective effect of heme oxygenase induction in ischemic acute renal failure. Crit Care Med 28: 809-817
Shiraishi, F., Curtis, L.M., Truong, L., Poss, K., Visner, G.A., Madsen, K., Nick, H.S., Agarwal, A., 2000. Heme oxygenase-1 gene ablation or expression modulates cisplatin-induced renal tubular apoptosis. Am J Physiol Renal Physiol 278: F726-F736
Shihab, F.S., 2002. Angiotensin II regulation of vascular endothelial growth factor and receptors Flt-1 and KDR/Flk-1 in cyclosporine nephrotoxicity. Kidney Int 62: 422–433
Singh, R.P., Patarca, R., Schwartz, J., Singh, P., Cantor, H., 1990. Definition of a specific interaction between the early T lymphocyte activation 1 (Eta-1) protein and murine macrophages in vitro and its effect upon macrophages in vivo. J Exp Med 171: 1931-1942
Stafford, N.P., Pink, A.E., White, A.E., Glenn, J.R., Heptinstall, S., 2003. Mechanisms involved in adenosine triphosphate--induced platelet aggregation in whole blood. Arterioscler Thromb Vasc Biol 23: 1928-1933
Stahl, R.A., Thaiss, F., Haberstroh, U., Kahf, S., Shaw, A., Schoeppe, W., 1990. Cyclooxygenase inhibition enhances rat interleukin 1 beta-induced growth of rat mesangial cells in culture. Am J Physiol 259: F419-424
Su, B., Karin, M., 1996. Mitogen activated protein kinase cascades and regulation of gene expression. Curr Opin Immunol 8: 402–411
Thomas, S.E., Lombardi, D., Giachelli, C., Bohle, A., Johnson, R.J., 1998. Osteopontin expression, tubulointerstitial disease, and essential hypertension. Am J Hypertens 11: 954-961
Taal, M.W., Zandi-Nejad, K., Weening, B., Shahsafaei, A., Kato, S., Lee, K.W., Ziai, F., Jiang, T., Brenner, B.M., MacKenzie, H.S., 2000. Proinflammatory gene expression and macrophage recruitment in the rat remnant kidney. Kidney Int 58: 1664-1676
Takahashi, T., Morita, K., Akagi, R., Sassa, S., 2004. Heme oxygenase-1: a novel therapeutic target in oxidative tissue injuries. Curr Med Chem 11: 1545-1561
Trachtman, H., Futterweit, S., Singhal, P.C., Sankaran, R., Franki, N., 1996. Renal tubular epithelial cell-E. coli interaction products stimulate nitric oxide production in cultured rat renal medullary interstitial and mesangial cells. Res Commun Mol Pathol Pharmacol 94: 227–238
Tsai, T.J., Lin, R.H., Chang, C.C., Chen, Y.M., Chen, C.F., Ko, F.N., Teng, C.M., 1995. Vasodilator agents modulate rat glomerular mesangial cell growth and collagen synthesis. Nephron 70: 91-99
Tsukinoki, T., Sugiyama, H., Sunami, R., Kobayashi, M., Onoda, T., Maeshima, Y., Yamasaki, Y., Makino, H., 2004. Mesangial cell Fas ligand: upregulation in human lupus nephritis and NF-kappaB- mediated expression in cultured human mesangial cells. Clin Exp Nephrol 8: 196-205
Ushio-Fukai, M., 1999. Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells. J Biol Chem 274: 22699–22704
Veis, J.H., 1993. An overview of mesangial cell biology. Contrib Nephrol 104: 115-126
Vijayakrishnan, L., Rudra, S., Eapen, M.S., Dastidar, S., Ray, A., 2007. Small-molecule inhibitors of PDE-IV and -VII in the treatment of respiratory diseases and chronic inflammation. Expert Opin Investig Drugs 16: 1585-1599
Vogt, B.A., Shanley, T.P., Croatt, A., Alam, J., Johnson, K.J., Nath, K.A., 1996. Glomerular inflammation induces resistance to tubular injury in the rat. A novel form of acquired, heme oxygenase-dependent resistance to renal injury. J Clin Invest 98: 2139-2145
Wang, Y, Rangan, G. K., Goodwin, B., Tay Y.C., Wang, Y., Harris, C.H., 2000. Lipopolysaccharide-induced MCP-1 gene expression in rat tubular epithelial cells is nuclear factor- B dependent. Kidney Int 57: 2011–2022
Weyrich, A.S., Denis, M.M., Luhlmann-Eyre, J.R., 2005. Dipyridamole selectively inhibits inflammatory gene expression in platelet-monocyte aggregates. Circulation 111: 632-642
Wiggins, K.J., Tiauw, V., Zhang, Y., Gilbert, R.E., Langham, R.G., Kelly, D.J., 2008. Perindopril attenuates tubular hypoxia and inflammation in an experimental model of diabetic nephropathy in transgenic Ren-2 rats. Nephrology 13(8):721-729
Williams, B., 1998. A potential role for angiotensin II-induced vascular endothelial growth factor expression in the pathogenesis of diabetic nephropathy. Miner Electrolyte Metab. 24: 400–405
Wolf, G., 1998. Angiotensin II is involved in the progression of renal disease: implication of non-hemodynamic mechanisms. Nephrologie 19: 451–456
Xie, Y., Sakatsume, M., Nishi, S., Narita, I., Arakawa, M., Gejyo, F., 2001. Expression, roles, receptors, and regulation of osteopontin in the kidney. Kidney Int 60: 1645-1657
Yoo, K.H., Thornhill, B.A., Forbes, M.S., Coleman, C.M., Marcinko, E.S., Liaw, L., Chevalier, R.L., 2006. Osteopontin regulates renal apoptosis and interstitial fibrosis in neonatal chronic unilateral ureteral obstruction. Kidney Int 70: 1735-1741
Young, B.A., Burdmann, E.A., Johnson, R.J., Alpers, C.E., Giachelli, C.M., Eng, E., Andoh, T., Bennett, W.M., Couser, W.G., 1995. Cellular proliferation and macrophage influx precede interstitial fibrosis in cyclosporine nephrotoxicity. Kidney Int 48: 439-448
Zatz, R., 2002. Mechanisms of progressive renal disease: role of angiotensin II, cyclooxygenase products and nitric oxide. J Hypertens 20: S37–S44
Zhou, Z., Song, R., Fattman, C.L., Greenhill, S., Alber, S., Oury, T.D., Choi, A.M., Morse, D., 2005. Carbon monoxide suppresses bleomycin-induced lung fibrosis. Am J Pathol 166: 27-37

------------------------------------------------------------------------ 第 9 筆 ---------------------------------------------------------------------
系統識別號 U0007-1901201011524700
論文名稱(中文) 慢性中耳炎病人耳膜破孔邊緣的組織病理學研究
論文名稱(英文) The histopathological study in the edge of perforated eardrum in patients with chronic otitis media
校院名稱 臺北醫學大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Graduate Institute of Clinical Medicine
學年度 98
學期 1
出版年 99
研究生(中文) 黃鈞鼎
學號 M102094032
學位類別 碩士
語文別 中文
口試日期 2009-12-24
論文頁數 60頁
口試委員 指導教授-李飛鵬
委員-李學禹
委員-賴銘堂
關鍵字(中) 中耳炎
組織病理學
關鍵字(英) otitis media
histopathology
muco-epithelial junction
學科別分類
中文摘要 中文摘要

研究背景及動機
慢性中耳炎病人的耳膜破孔,大多不會自然癒合,其原因尚未完全被了解。一般認為是由於耳膜破孔邊緣的耳膜外層上皮及內層上皮形成連合(muco-epithelial junction, MEJ)所致。但在臨床的觀察,慢性中耳炎病人耳膜破孔除了反覆感染或耳漏之外,我們常會見到所謂永久性破孔,在不同的時間,會有不同的型態變化;有的形成新的肉芽組織,有的呈現角質上皮移行,有的破孔甚至會自行癒合。過去所知的「永久」的瘢痕性破孔,或是MEJ造成的的癒合作用停滯,似乎不足以解釋這些臨床變化。本研究希望藉觀察耳膜破孔邊緣的組織病理學,進一步探討影響耳膜癒合的因素。

研究材料與方法
本研究目前共收集43例慢性中耳炎合併中心型耳膜破孔的病人檢體,將鼓室成形術中耳膜破孔邊緣切除的組織標本,作成系列組織切片,以Hematoxylin、Eosin、cytokeratin 7及19免疫抗體染色,以及Collagen type I與III免疫抗體染色,觀察耳膜破孔邊緣MEJ的分布,組織形態的變化。並追蹤病人的手術後耳膜的表皮移行完整程度,再與臨床表現及病理組織觀察等變項互相印證。

結果
本研究的43個案例中,19例為男性,24例為女性,年齡介於到20歲到77歲之間,平均為45歲。其中年輕組有15例,年長組有28例。術後表皮移行完整的有36例,不完整的有7例。其中有28例的MEJ在檢體的系列切片觀察不到,有5例檢體具有連續的的MEJ,有10例檢體的MEJ在檢體的系列切片見到不連續的分布。Cytokeratin 7及19免疫抗體染色大多數的結果一致,染在切片中耳膜內側的mucosa層,但有10例檢體的Cytokeratin 19更出現在麟狀上皮的基底層;Collagen type I與III免疫抗體染色也顯示,在不同的傷口癒合狀態下,Collagen type I與III的分布多寡會有差異。各變項與術後表皮移行完整之間,年齡分組具有顯著差異( p=0.037),其餘有collagen type III/I ratio (p=0.02)及MEJ (p=0.047)達顯著差異,經多變項回歸檢視後,對術後表皮移行完整與否,也只有collagen III/I ratio及MEJ足以達到顯著差異(p=0.007)。與年齡分組呈顯著相關的有術後表皮移行完整與否( p=0.037)、collagen type III/I ratio (p=0.0)及wound healing stage等,而其餘各個變項未達顯著程度。

結論
本研究的結果顯示慢性中耳炎耳膜破孔邊緣MEJ的變化,即表皮在耳膜破孔邊緣的變化,和纖維層Collagen type III/I比例,會影響術後的表皮移行完整程度。而MEJ的位置,並非一成不變的存在於耳膜破孔邊緣,至少與先前的研究結果有所差異。同時本研究也發現有10個案例的複層麟狀上皮,在基底層對CK-19抗體呈濃染,這也是第一次在人類耳膜組織的研究中,證實耳膜破孔邊緣也有幹細胞的存在。這些結果應具有更進一步研究及應用的價值。



















英文摘要 Abstract

Introduction
Eardrum perforation in patients with chronic otitis media results from failure in spontaneous healing process when it ruptured. The hypothesis of muco-epithelail junction (MEJ) formation over the perforation edge is widely accepted to explain the persistent pathophysiological state. However, clinically there are still dynamic changes noted in some chronic perforations. The aim of this study is to investigate the possible factors engaged in healing process of perforated eardrum from chronic otitis media through observation of the histopathological sections.

Material and Methods
In our study, 43 specimens from the eardrum of chronic otitis media were enrolled. Series section and immunohistochemical stain with Hemotoxylin, Eosin, Cytokeratin 7 and 19 antibody, and Collagen type I and III antibody were performed each case. Statistic analysis was done to compare these variables with the postoperative epithelial migration ability.


Result
There were 43 cases enrolled in this study. Nineteen cases were male and 24 cases were female. The age ranged from 20 to 77 years with average 45 years. Fifteen cases were in the young group; twenty-eight cases were in the aged group. Thirty-six cases showed complete postoperative re-epithelialization of squamous epithelium on the eardrum surface and others not. Histopathological examination revealed 28 cases without MEJ, 5 cases with continuous MEJ, and 8 cases with interrupted MEJ. The basal layer of squamous epithelium was positive stained with CK-19 in 10 cases. Collagen type I and III showed different in distribution with different wound healing stage. Statistically, age group ( p=0.037), collagen type III/I ratio (p=0.02) and MEJ (p=0.047) showed a significant correlation with the postoperative re-epithelialization of squamous epithelium. However, multiple linear regression model showed only the MEJ and collagen type III/I ratio significant correlated with the postoperative re-epithelialization of squamous epithelium (p=0.007). On the other hand, collagen type III/I ratio (p=0.0) and wound healing stage were also significant correlated with the age group.


Conclusions:
The result in this study showed a significant correlation between the MEJ distribution, collagen type III/I ratio and the postoperative re-epithelialization of squamous epithelium. And the MEJ distribution in our study is quite different from the previous studies. The 10 cases, with CK-19 positive stained basal layer of squamous epithelium, showed an evidence of stem cells in the perforated edge in patients with chronic otitis media in a very first time. These results may contribute to further investigations and studies in this field.















論文目次 目錄

中文摘要 i
英文摘要(Abstract) iv
第一章 緒論 1
第二章 文獻探討 3
壹、慢性中耳炎 3
第一節 定義及流行病學 4
第二節 臨床症狀 5
第三節 病因學與發生機轉 7
第四節 常見併發症 10
第五節 診斷 11
第六節 治療及預後 12
第七節 修補耳膜破孔的主要手術方式 14
貳、耳膜破孔的癒合 15
第一節 耳膜的組織學 15
第二節 耳膜破孔癒合的機轉 17
第三節 永久性破孔的成因 20
參、細胞角蛋白 22
第一節 細胞角蛋白概論 22
第二節 細胞角蛋白在耳膜組織學的相關文獻 24
肆、細胞外間質與傷口復原性的關連性 25
第三章 研究動機與目的 26
第四章 研究方法 28
壹、研究對象及檢體採集 28
第一節 研究對象 28
第二節 手術方式與檢體採集 29
第三節 標本備製與切片方法 30
貳、實驗材料、儀器及實驗步驟 31
第一節、材料與染劑 31
第二節、Hematoxylin-Eosin染色 32
第三節、Cytokeratin antibody免疫組織化學染色 33
第四節、Collagen antibody免疫組織化學染色 35
第五節、觀察與記錄 37
參、檢體分組與結果判定 38
肆、統計方法 39
第五章 結果分析 40
壹、基本資料 40
貳、組織學觀察 42
第一節、HE染色與CK-7與CK-19抗體染色 42
第二節、Collagen type I、III免疫組織化學染色 46
參、統計分析結果 49
第六章 討論 53
第七章 結論 58
參考文獻 59
參考文獻 1. Somers, T., et al., Histology of the perforated tympanic membrane and its muco-epithelial junction. Clin Otolaryngol Allied Sci, 1997. 22(2): p. 162-6.
2. Boedts, D. and B. Ars, Histopathological research on eardrum perforations. Arch Otorhinolaryngol, 1977. 215(1): p. 55-9.
3. Berger, G., Nature of spontaneous tympanic membrane perforation in acute otitis media in children. J Laryngol Otol, 1989. 103(12): p. 1150-3.
4. Graham, M.D., A Longitudinal Study of Ear Disease and Hearing Loss in Patients with Cleft Lips and Palates. Ann Otol Rhinol Laryngol, 1964. 73: p. 34-47.
5. Soudijn, E.R. and A.J. Huffstadt, Cleft palates and middle ear effusions in babies. Cleft Palate J, 1975. 12: p. 229-33.
6. Karma, P., Middle ear epithelium and chronic ear disease. Acta Otolaryngol Suppl, 1972. 307: p. 1-107.
7. O'Neill, P., Acute otitis media. Clin Evid, 2002(8): p. 251-61.
8. Lim, D.J., Tympanic membrane. Electron microscopic observation. I: pars tensa. Acta Otolaryngol, 1968. 66(3): p. 181-98.
9. Broekaert, D., The tympanic membrane: a biochemical updating of structural components. Acta Otorhinolaryngol Belg, 1995. 49(2): p. 127-37.
10. Weinberger, J.M. and M. Hawke, Thickened patches of stratum corneum on the human tympanic membrane. J Otolaryngol, 1986. 15(6): p. 327-31.
11. Kakoi, H. and M. Anniko, Auditory epithelial migration. II: Morphological evidence for auditory epidermal cell migration in rat. Acta Otolaryngol, 1996. 116(6): p. 850-3.
12. Makino, K., et al., Epithelial migration in the healing process of tympanic membrane perforations. Eur Arch Otorhinolaryngol, 1990. 247(6): p. 352-5.
13. Schmidt, S.H. and S. Hellstrom, Late effects of local anesthetics on tympanic membrane structure. Am J Otolaryngol, 1986. 7(5): p. 346-52.
14. Johnson, A. and M. Hawke, The function of migratory epidermis in the healing of tympanic membrane perforations in guinea-pig. A photographic study. Acta Otolaryngol, 1987. 103(1-2): p. 81-6.
15. Spandow, O., S. Hellstrom, and M. Dahlstrom, Structural characterization of persistent tympanic membrane perforations in man. Laryngoscope, 1996. 106(3 Pt 1): p. 346-52.
16. Rizer, F.M., Overlay versus underlay tympanoplasty. Part I: historical review of the literature. Laryngoscope, 1997. 107(12 Pt 2): p. 1-25.
17. Vennix, P.P., et al., Epidermal differentiation in the human external auditory meatus. Laryngoscope, 1996. 106(4): p. 470-5.
18. Koba, R., Epidermal cell migration and healing of the tympanic membrane: an immunohistochemical study of cell proliferation using bromodeoxyuridine labeling. Ann Otol Rhinol Laryngol, 1995. 104(3): p. 218-25.
19. Govaerts, P.J., W.A. Jacob, and J. Marquet, Histological study of the thin replacement membrane of human tympanic membrane perforations. Acta Otolaryngol, 1988. 105(3-4): p. 297-302.
20. Ramalho, J.R. and R.F. Bento, Healing of subacute tympanic membrane perforations in chinchillas treated with epidermal growth factor and pentoxifylline. Otol Neurotol, 2006. 27(5): p. 720-7.
21. Ishibashi, T., et al., Induction of KGF, basic FGF, and TGFalpha mRNA expression during healing of experimental TM perforations. Acta Otolaryngol, 1998. 118(5): p. 701-4.
22. Bombardieri, E., et al., Evaluation of cytokeratin 19 serum fragments (CYFRA 21-1) in patients with lung cancer: results of a multicenter trial. Int J Biol Markers, 1994. 9(2): p. 89-95.
23. Welkoborsky, H.J., et al., Molecular biologic characteristics of seven new cell lines of squamous cell carcinomas of the head and neck and comparison to fresh tumor tissue. Oncology, 2003. 65(1): p. 60-71.
24. Broekaert, D., et al., Immunohistochemical analysis of the cytokeratin expression in middle ear cholesteatoma and related epithelial tissues. Ann Otol Rhinol Laryngol, 1992. 101(11): p. 931-8.
25. Lepercque, S., D. Broekaert, and P. Van Cauwenberge, Cytokeratin expression patterns in the human tympanic membrane and external ear canal. Eur Arch Otorhinolaryngol, 1993. 250(2): p. 78-81.
26. Kim, H.J., S.P. Tinling, and R.A. Chole, Expression patterns of cytokeratins in cholesteatomas: evidence of increased migration and proliferation. J Korean Med Sci, 2002. 17(3): p. 381-8.
27. Kasper, M., Patterns of cytokeratin and vimentin expression in the human eye. Histochemistry, 1988. 89: p. 9.
28. Wang, W.Q., Z.M. Wang, and J. Tian, [Epidermal stem cells in the tympanic membrane]. Zhonghua Er Bi Yan Hou Ke Za Zhi, 2004. 39(12): p. 712-6.
29. Byung-Moo, K.S.R.J.L.L.G.M., Electrospinning of collagen nanofibers : Effects on the behavior of normal human keratinocytesand earlysatge wound healing. Biomaterials, 2006. 27(8): p. 10.
30. Liang, J., L. Michaels, and A. Wright, Immunohistochemical characterization of the epidermoid formation in the middle ear. Laryngoscope, 2003. 113(6): p. 1007-14.




------------------------------------------------------------------------ 第 10 筆 ---------------------------------------------------------------------
系統識別號 U0007-2107200921451500
論文名稱(中文) 原住民族群(Atayal ,泰雅)URAT1蛋白對應GENE SLC22A12上rs893006 SNP位點於高尿酸血症患者中之表現
論文名稱(英文) Association between rs893006 SNP of gene SLC22A12 encoding URAT1 (urate transporter protein) and the hyperuricemia in Taiwanese aborigines (Atayal)
校院名稱 臺北醫學大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Graduate Institute of Clinical Medicine
學年度 97
學期 2
出版年 98
研究生(中文) 石瑄
學號 M102092025
學位類別 碩士
語文別 中文
口試日期 2009-06-27
論文頁數 53頁
口試委員 委員-蔡清霖
委員-陳中庸
委員-朱絹秀
委員-何為斌
指導教授-謝銘勳
關鍵字(中) 痛風
尿酸
單核苷
酸多態性
台灣原住民
關鍵字(英) gout
uric acid
single nucleotide polymorphism
taiwanese aborigines
學科別分類
中文摘要 尿酸是人體嘌呤代謝後的最終產物(ATP, GTP & nucleic acid), 而人體的尿酸經由製造和排出維持一定的濃度.當尿酸在體內的濃度過高時, 會很容易累積在關節周圍, 刺激發炎,腫脹,發熱, 引起嚴重的疼痛, 甚至久了會形成痛風石, 破壞關節組織, 包括肌腱及骨頭部份.當疼痛發生時, 造成相當大的痛苦, 有人形容成風吹過也疼痛不已, 故也叫做痛風.
一直以來, 尿酸經由何種模式代謝, 其最終對人體是不是無任何幫助, 只會產生疾病, 尚未有定論. 但已知的是尿酸對人體而論, 似乎不是一無是處. 在一些特定情況下, 尿酸可以是一種強的抗氧化物,當缺乏鹽類時, 還可以經由血管收縮素做用, 扮演穩定血壓的重要角色.
近端腎小管頂點蛋白URAT1(coded by SLC22A12), 對尿酸經由腎臟的排出及再吸收所扮演的角色, 重要性似乎是愈來愈明顯, 而陸續有一些新的報告針對SLC22A12其核酸多樣性對尿酸代謝的影響關係被定性出來.經由326位日本人之SLC22A12 gene, 點rs893006單核苷酸多態性(single neucleotide polymorphism, SNP) (GG, GT and TT) 之分析, 發現血液中高尿酸值與SLC22A12 基因中之單核苷酸多態性有明顯相關.
在台灣原住民中, 有明顯的高尿酸血症之發生率. 我們採取368位志願者之血液, 包括175位男性受試者及193位女性受試者. 分別為原住民235位(泰雅族、布農族、排灣族)和平地人133位. 淋巴球純化出DNA, 檢視SLC22A12 gene點rs893006單核苷酸多態性(single neucleotide polymorphism, SNP)的基因表現型為何, 其不同基因型對於URAT1蛋白的功能影響與高尿酸血症有何影響, 其關係為何, 是否有意義?
另外, 我們也將同時比較血液樣本中之肌酸酐, 空腹血糖值, 三酸甘油脂, 胆固醇, 基礎體重值與高尿酸血症患者其SLC22A12 gene點rs893006單核苷酸多態性是否有任何相關性.
英文摘要 Serum uric acid is the degradation product of purines (ATP, GTP & nucleic acid). Serum uric acid level is maintained by urate synthesis and excretion. Whenever hyperurecemia happened, the joint inflammatory change, cause a lot of pain. The risk of deposition of uric acid around joints will increase, thus cause joint, tendon destruction and disability. The tophus formation and joint destructive process induce very severe pain and disability, which is known as gouty arthritis.
For a long time, uric acid metabolitic process is not been fully understood. Is it really the only final excretional product or if it still has some possible usefulness? The answer is quite clear, under some certain circumstances, it can be function similar to vitamin C, as a potent antioxidant. Also, urate can maintain blood pressure under low salt conditions via stimulation of the reninangiotensin system through a mechanism that is still poorly understood.
The renal tubule apical protein, URAT1 (coded by SLC22CA12) was recently proposed to be the major absorptive urate transporter protein in the kidney regulating blood urate levels. A study of the Janpanese genetic variations in SLC22A12 gene, rs893006 polymorphism (GG, GT and TT) in a total of 326 Japanese subjects was published. The significant correlation between single nucleotide polymorphism (SNP) in the urate transporter gene SLC22CA12 was found to be associated with the elevated serum uric acid levels.
In Taiwanese aborigines, has a remarkably high prevalence of hyperuricemia and gout. We collected 368 volunteers blood samples, which including 175 cases of male and 193 cases of female. (Ataya, Bunun, Paiwan, and general Taiwanese)(including 133 cases of general population of taiwanese as control group, 235 cases are the Taiwanese aborigines(Atayal),) The genomic DNA from peripheral blood lymphocytes will be collected, and use for genotyping of the rs893006 polymorphism in SLC22A12 gene, comparing the difference between Taiwanese aborigines and the general population.
Otherwise, we will also compare the difference between the plasma level of creatinine, fasting plasma glucose level, triglyceride, serum cholesterol, BMI and the serum uric acid level.
論文目次 誌謝 02
圖表目次 04
章節目錄 05
中文摘要 06
英文摘要 08
縮寫表
緒論 11
研究方法與材料 18
結果 24
討論 31
結論 37
参考文獻 38
圖表
參考文獻 1. Anzai N et al. The multivalent PDZ domain-containing protein PDZK1 regulates transport activity of renal urate-anion exchanger URAT1 via its C terminus.
J Biol Chem 279: 45942–45950
2. B Stibůrková Familial juvenile hyperuricemic nephropathy: Localization of the gene on chromosome 16p11.2—and evidence for genetic heterogeneity
Am. J. Hum. Genet. 66:1989–1994, 2000
3. Cappuccio, F.P Uric acid metabolism and tubular sodium handling.
JAMA 270, 354–359
4. Choi HK, Mount DB, Reginato AM Pathogenesis of gout.
Ann Intern Med 143: 499–516
5. C. T. CHOU The epidemiology of hyperuricemia and gout in Taiwan aborigines
British Journal of Rheumatology 1998;37:258–262
6. Dehghan A et al. Association of three genetic loci with uric acid concentration and risk of gout: a genome-wide association study.
Lancet 372: 1953–1961.
7. Doring A, Gieger C et al. SLC2A9 influences uric acid concentrations with pronounced sex-specific effects.
Nat Genet 40: 430–436.
8. Endou, H Gene analysis of urate transporter gene, URAT1. Joint Research
Report in Kyorin University
9. Enomoto A, Kimura H et al Molecular identification of a renal urate anion exchanger that regulates blood urate levels.
Nature 417: 447–452, 2002
10. Fredriksson R The solute carrier (SLC) complement of the human genome: phylogenetic classification reveals four major families.
FEBS Lett 582: 3811–3816.
11. George Nuki and Peter A Simkin A concise history of gout and hyperuricemia and their treatment
Arthritis Research & Therapy 2006, 8(Suppl 1)
12. Horikawa, Y et al Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus.
Nature Genetics 26, 163–175.
13. Ichida, K. et al Age and origin of the G774A mutation in SLC22A12 causing renal hypouricemia in Japanese.
Clin. Genet. 74: 243-251, 2008
14. Iwai, N., Mino, Y High prevalence of renal hypouricemia caused by inactive SLC22A12 in Japanese.
Kidney Int. 66, 935–944
15. Jang, Won Cheoul et al T6092C polymorphism of SLC22A12 gene is associated with serum uric acid concentrations in Korean male subjects
Clin Chim Acta. 2008 Dec:398(1-2):140-4. Epub 2008 Sep
16. Juergen Graessler Association of the human urate transporter 1 with reduced renal uric acid excretion and hyperuricemia in a German Caucasian population
Arthritis & rheumatism 2006 Jan;54(1):292-300
17. J Vázquez-Mellado Homozygous frameshift mutation in the SLC22A12 gene in a patient with primary gout and high levels of serum uric acid
Journal of Clinical Pathology 2007;60:947-948
18. Kagan, A Epidemiologic studies of coronary heart disease and stroke in Japanese men living in Japan, Hawaii and California: demographic, physical, dietary and biochemical characteristics.
Journal of Chronic Diseases 27, 345–364.
19. Kikuchi Y et al Patients with renal hypouricemia
with exercise-induced acute renal failure and chronic
renal dysfunction.
Clin Nephrol 53:467–472, 2000
20. Kimiyoshi ichida, Makoto hosoyamada Clinical and molecular analysis of patients with renal hypouricemia in Japan-Influence of URAT1 gene on urinary urate excretion J Am Soc Nephrol 15: 164–173, 2004
21. Lesch, M., Nyhan, W.L., 1964. A family disorder of uric acid metabolism and central nervous system function.
American Journal of Medicine 36, 561–570.
22. Li Shu-Chuan Cheng Genomewide scan for gout in Taiwanese aborigines reveals linkage to chromosome 4q25 Am. J. Hum. Genet. 75:498–503, 2004
23. Matthias A Molecular physiology of urate transport Physiology. Volume 20, April 2005
24. M. Guan High-resolution melting analysis for the rapid detection of an intronic single nucleotide polymorphism in SLC22A12 in male patients with primary gout in China
Scandinavian journal of rheumatology, Volume 38, Issue 4 2009, pages 1-6
25. Melanie Kolz Meta-analysis of 28,141 individuals identifies common variants within five new loci that influence uric acid concentrations
PLoS Genet. 2009 June; 5(6): e1000504
26. Michael H. Pillinger Hyperuricemia and gout Bulletin of the NYU Hospital for Joint Diseases 2007;65(3):215-21
27. Rosa J. Torres Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency: Lesch-Nyhan syndrome
Orphanet Journal of Rare Diseases 2007, 2:48 doi:10.1186/1750-1172-2-48
28. Siguang Li The GLUT9 gene is associated with serum uric acid levels in Sardinia and Chianti Cohorts
PLoS Genet. 2007 November; 3(11): e194
29. S. Sutaria Effectiveness of interventions for the treatment of acute and prevention of recurrent gout—a systematic review
Rheumatology 2006;45:1422–1431
30. Taniguchi A, Kamatani N Control of renal uric acid excretion and gout.
Curr Opin Rheumatol 20: 192–197.
31. Tzovaras V et al Absence of SLC22A12 gene mutations in Greek Caucasian patients with primary renal hypouricaemia
Scand J Clin Lab Invest. 2007;67(6):589-95
32. Vázquez-Mellado J Molecular analysis of the SLC22A12 (URAT1) gene in patients with primary gout
Rheumatology 2007 Feb;46(2):215-9. Epub 2006 Jul 11
33. Whitfield JB, Martin NG Inheritance and alcohol as factors influencing plasma uric acid levels.
Acta Genet Med Gemellol (Roma ) 32: 117–126.
34. Yukio Shima a Association between intronic SNP in urate-anion exchanger gene, SLC22A12, and serum uric acid levels in Japanese
Life Sciences 79 (2006) 2234–2237
35. Zdenek Dvorak et al Cytotoxictiy of colchicines derivatives in primary cultures of human hepatocytes
Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2007, 151(1):47–52

------------------------------------------------------------------------ 第 11 筆 ---------------------------------------------------------------------
系統識別號 U0007-2201201001482700
論文名稱(中文) 化學治療誘發之胰臟間質細胞早發老化對胰臟腺癌復發及抗藥性之異源性影響
論文名稱(英文) The Heterotypic Influences on Chemotherapeutic Agent-induced Prematurely Senescent Stromal Cells on the Malignant Progression of Pancreatic Adenocarcinoma
校院名稱 臺北醫學大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Graduate Institute of Clinical Medicine
學年度 98
學期 1
出版年 99
研究生(中文) 張智翔
學號 M118096003
學位類別 碩士
語文別 英文
口試日期 2009-12-30
論文頁數 78頁
口試委員 委員-王育民
委員-李宗儒
委員-賴文福
共同指導教授-蔡坤志
指導教授-連吉時
關鍵字(中) 胰臟癌
基質
微環境
星狀細胞
化學治療
細胞老化
關鍵字(英) pancreatic cancer
stroma
microenvironments
stellate cells
chemotherapy
senescence.
學科別分類
中文摘要 背景:腫瘤基質的微環境 (Microenvironment) 會極度影響上皮腫瘤發生的許多步驟。胰臟星狀細胞 (Pancreatic stellate cells) 是造成胰臟腺癌腫瘤纖維化基質 (Desmoplastic stroma) 微環境的主要原因。胰臟星狀細胞和腫瘤細胞間的異源交互作用 (Heterotypic interaction) 在腫瘤的惡性進展 (Malignant progression) 過程也扮演著重要的角色。最近的研究發現,胰臟星狀細胞會被因暴露於低於致死的傷源(如游離輻射及細胞毒性之化學治療藥物)後被誘發進行類似於細胞老化 (Cell senescence) 的變化。更有趣的是,表現這種壓力誘發性早發細胞老化 (Stress-induced premature senescence) 的細胞也會藉由分泌蛋白及細胞外蛋白來對鄰近細胞產生旁分泌作用。延續這種說法,壓力誘發性早發細胞老化之基質纖維細胞也被證實能夠促進多種人類腺體腫瘤的成長及侵犯。

目的:我們假設壓力誘發性早發老化之胰臟星狀細胞在接受臨床使用之化學治療藥物劑量後會累積於胰臟的基質組織,進而逐漸形成一個允許胰臟癌產生抗藥及覆發之惡性腫瘤進展的允許微環境 (Permissive microenvironments)。

方法和結果:在臨床使用劑量下,胰臟星狀細胞確實可以藉由不同種類的細胞毒性化學治療藥物導致壓力誘發之早發老化之特質。在所有的藥物中,Gemcitabine在其臨床使用劑量(10μM x 30分鐘)最能有效地促成早發老化之表現。利用基因體的分析方式,我們發現壓力誘發性早發老化之胰臟星狀細胞能夠調增和壓力、傷口癒合及細胞凋零相關基因的表現。更重要的是,壓力誘發性早發老化之胰臟星狀細胞更會製造多種和細胞素卅趨化素之訊息傳遞及細胞間質重塑相關之蛋白。我們也進一步的利用三次元細胞共同培養之模式證明了早發老化之胰臟星狀細胞對腫瘤惡性進展的促成。為了進一步證實活體外之發現,當被和胰臟癌細胞同時被植入有免疫缺陷的老鼠時,早發老化之胰臟星狀細胞確實能促進腫瘤的發展。
結論:我們的觀察發現,當胰臟星狀細胞受到細胞毒性化學藥物的反覆暴露後,特別是gemcitabine,星狀細胞可以被誘發類似老化之顯性特徵。壓力誘發性早發老化之胰臟星狀細胞不管在活體外或體內的條件之下均可產生一個類似於傷口癒合過程且有利於腫瘤發展的微允許環境。胰臟星狀細胞和胰癌細胞之間的異源交互作用或許是胰臟癌在治療抗性之機轉,同時也可能成為未來潛在的治療靶標。
英文摘要 Background: The stromal microenvironments in which tumors develop profoundly influence many steps of epithelial tumorigenesis. Pancreatic stellate cells (PaSCs) are the major contributors of the desmoplastic stromal microenvironment of pancreatic adenocarcinoma and play a crucial role in malignant progression through their heterotypic interactions with tumor cells. Recent evidence suggests that cells may develop a senescent-like growth arrest program when they are exposed to sub-lethal injuries such as ionizing radiation and/or cytotoxic agents. Intriguingly, cells displaying this stress-induced premature senescence (SIPS) phenotype can elicit paracrine signaling through their induced production of a variety of secretory factors and extracellular proteins. Along this line, SIPS stromal fibroblasts have been shown to promote tumor growth and invasion in several types of human glandular malignancies,

Aim: We hypothesize that SIPS PaSCs may accumulate in the pancreatic stroma over time following cytotoxic chemotherapies at a clinically relevant manner and which may gradually create more permissive microenvironments for the ensuing malignant progression of treatment-resistant or relapsing pancreatic cancers.

Result: A SIPS phenotype was induced in PaSCs by different cytotoxic chemotherapeutic agents that have been used clinically for the treatment of pancreatic cancer. Among them, gemcitabine was most effective in the induction of the SIPS phenotype, which occurred at clinically relevant concentrations (10 μM for 30 minutes). Using genomic profiling approach, we show that SIPS PaSCs upregulated the expressions of several groups of genes involved in stress and wound response, and apoptosis. Most importantly, SIPS PaSCs secreted proteins involved in cytokine/chemokine signaling and extracellular matrix remodeling. Using an in vivo-like three dimensional culture system, we further demonstrated that SIPS PaSCs promoted the invasive growth of co-cultivated pancreatic cancer cells. Corroborating the in vitro findings, SIPS PaSCs, when co-implanted with pancreatic cancer cells subcutaneously into immunocompromised mice, can significantly promoted the growth of xenografted pancreatic tumors.

Conclusion: Our observations suggest that repeatitive exposure of PaSCs to cytotoxic chemotherapeutic agents, especially gemcitabine, induces phenotypical changes in PaSCs resembling senescence. SIPS PaSCs can create a permissive microenvironment similar to that created during the wound healing process and promote tumor progression both in vitro and in vivo. The heterotypic interactions between SIPS PaSCs and pancreatic carcinoma cells may shed a new light on the mechanistic basis of treatment refractoriness in pancreatic adenocarcinoma and may serve as potential therapeutic targets thereof.
論文目次 Abstract in Chinese..........i
Abstract in English.........iv
Introduction.................1
Hypothesis..................17
Materials and Methods.......19
Results.....................27
Discussion..................35
Future Work.................46
Reference ...................53
Figure......................64
參考文獻 1. Jemal, A., et al., Cancer statistics, 2008. CA Cancer J Clin, 2008. 58(2): p. 71-96.
2. Lillemoe, K.D., C.J. Yeo, and J.L. Cameron, Pancreatic cancer: state-of-the-art care. CA Cancer J Clin, 2000. 50(4): p. 241-68.
3. Gemmel, C., et al., Pancreatic cancer screening: state of the art. Expert Rev Gastroenterol Hepatol, 2009. 3(1): p. 89-96.
4. Yeo, T.P., et al., Pancreatic cancer. Curr Probl Cancer, 2002. 26(4): p. 176-275.
5. Cullinan, S., et al., A phase III trial on the therapy of advanced pancreatic carcinoma. Evaluations of the Mallinson regimen and combined 5-fluorouracil, doxorubicin, and cisplatin. Cancer, 1990. 65(10): p. 2207-12.
6. Burris, H.A., 3rd, et al., Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol, 1997. 15(6): p. 2403-13.
7. Shepherd, F.A., et al., Gemcitabine in the treatment of elderly patients with advanced non-small cell lung cancer. Semin Oncol, 1997. 24(2 Suppl 7): p. S7-50-S7-55.
8. Heinemann, V., Gemcitabine plus cisplatin for the treatment of metastatic breast cancer. Clin Breast Cancer, 2002. 3 Suppl 1: p. 24-9.
9. Heinemann, V., Gemcitabine-based combination treatment of pancreatic cancer. Semin Oncol, 2002. 29(1 Suppl 3): p. 25-35.
10. Calvero., Chemical structure of gemcitabine (trade name gemzar). January 2007.
11. Sivalakshmidevi, A., et al., 2'-Deoxy-2',2'-difluorocytidine monohydrochloride (Gemcitabine hydrochloride) DRL publication No. 256. Acta Crystallographica Section E, 2003. 59(10): p. o1435-o1437.
12. Mackey, J.R., et al., Nucleoside transport and its significance for anticancer drug resistance. Drug Resist Updat, 1998. 1(5): p. 310-24.
13. Grunewald, R., et al., Saturation of 2',2'-difluorodeoxycytidine 5'-triphosphate accumulation by mononuclear cells during a phase I trial of gemcitabine. Cancer Chemother Pharmacol, 1991. 27(4): p. 258-62.
14. Abbruzzese, J.L., et al., A phase I clinical, plasma, and cellular pharmacology study of gemcitabine. J Clin Oncol, 1991. 9(3): p. 491-8.
15. Grunewald, R., et al., Gemcitabine in leukemia: a phase I clinical, plasma, and cellular pharmacology study. J Clin Oncol, 1992. 10(3): p. 406-13.
16. Chabner, B.A., et al., Chapter 51. Antineoplastic Agents" . Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11e.
17. Miura, T., et al., [A case of interstitial pneumonia induced by gemcitabine hydrochloride for unresectable bile duct cancer]. Gan To Kagaku Ryoho, 2009. 36(10): p. 1757-60.
18. Galvao, F.H., J.O. Pestana, and V.L. Capelozzi, Fatal gemcitabine-induced pulmonary toxicity in metastatic gallbladder adenocarcinoma. Cancer Chemother Pharmacol, 2010. 65(3): p. 607-10.
19. Glezerman, I., et al., Gemcitabine nephrotoxicity and hemolytic uremic syndrome: report of 29 cases from a single institution. Clin Nephrol, 2009. 71(2): p. 130-9.
20. Storniolo, A.M., et al., An investigational new drug treatment program for patients with gemcitabine: results for over 3000 patients with pancreatic carcinoma. Cancer, 1999. 85(6): p. 1261-8.
21. W. F. Regine, K.W.W., R. Abrams, H. Safran, J. P. Hoffman, A. Konski, A. B. Benson, J. S. MacDonald, C. G. Willett, T. A. Rich, RTOG 9704 a phase III study of adjuvant pre and post chemoradiation (CRT) 5-FU vs. gemcitabine (G) for resected pancreatic adenocarcinoma. Journal of Clinical Oncology, 2006. 24(18S): p. 4007.
22. Oettle, H., et al., Adjuvant chemotherapy with gemcitabine vs observation in patients undergoing curative-intent resection of pancreatic cancer: a randomized controlled trial. JAMA, 2007. 297(3): p. 267-77.
23. Berlin, J.D., et al., Phase III study of gemcitabine in combination with fluorouracil versus gemcitabine alone in patients with advanced pancreatic carcinoma: Eastern Cooperative Oncology Group Trial E2297. J Clin Oncol, 2002. 20(15): p. 3270-5.
24. Heinemann, V., et al., Randomized phase III trial of gemcitabine plus cisplatin compared with gemcitabine alone in advanced pancreatic cancer. J Clin Oncol, 2006. 24(24): p. 3946-52.
25. Rocha Lima, C.M., et al., Irinotecan plus gemcitabine results in no survival advantage compared with gemcitabine monotherapy in patients with locally advanced or metastatic pancreatic cancer despite increased tumor response rate. J Clin Oncol, 2004. 22(18): p. 3776-83.
26. Louvet, C., et al., Gemcitabine in combination with oxaliplatin compared with gemcitabine alone in locally advanced or metastatic pancreatic cancer: results of a GERCOR and GISCAD phase III trial. J Clin Oncol, 2005. 23(15): p. 3509-16.
27. Oettle, H., et al., A phase III trial of pemetrexed plus gemcitabine versus gemcitabine in patients with unresectable or metastatic pancreatic cancer. Ann Oncol, 2005. 16(10): p. 1639-45.
28. Cunningham, D., I. Chau, and D. Stocken, Phase III randomised comparison of gemcitabine (GEM) versus gemcitabine plus capecitabine (GEM-CAP) in patients with advanced pancreatic cancer. Eur J Cancer. 2005(3): p. 4.
29. Moore, M.J., et al., Erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer: a phase III trial of the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol, 2007. 25(15): p. 1960-6.
30. Kim, M.P. and G.E. Gallick, Gemcitabine resistance in pancreatic cancer: picking the key players. Clin Cancer Res, 2008. 14(5): p. 1284-5.
31. Xu, Z.W., et al., Overexpression of Bax sensitizes human pancreatic cancer cells to apoptosis induced by chemotherapeutic agents. Cancer Chemother Pharmacol, 2002. 49(6): p. 504-10.
32. Yin, T., et al., Expression of snail in pancreatic cancer promotes metastasis and chemoresistance. J Surg Res, 2007. 141(2): p. 196-203.
33. Miyamoto, H., et al., Tumor-stroma interaction of human pancreatic cancer: acquired resistance to anticancer drugs and proliferation regulation is dependent on extracellular matrix proteins. Pancreas, 2004. 28(1): p. 38-44.
34. Chang, H.Y., et al., Diversity, topographic differentiation, and positional memory in human fibroblasts. Proc Natl Acad Sci U S A, 2002. 99(20): p. 12877-82.
35. Hanahan, D. and R.A. Weinberg, The hallmarks of cancer. Cell, 2000. 100(1): p. 57-70.
36. Mollenhauer, J., I. Roether, and H.F. Kern, Distribution of extracellular matrix proteins in pancreatic ductal adenocarcinoma and its influence on tumor cell proliferation in vitro. Pancreas, 1987. 2(1): p. 14-24.
37. Apte, M.V., et al., Desmoplastic reaction in pancreatic cancer: role of pancreatic stellate cells. Pancreas, 2004. 29(3): p. 179-87.
38. Folkman, J., Tumor angiogenesis: therapeutic implications. N Engl J Med, 1971. 285(21): p. 1182-6.
39. O'Reilly, M.S., et al., Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell, 1997. 88(2): p. 277-85.
40. Folkman, J., et al., Isolation of a tumor factor responsible for angiogenesis. J Exp Med, 1971. 133(2): p. 275-88.
41. Ishii, G., et al., Bone-marrow-derived myofibroblasts contribute to the cancer-induced stromal reaction. Biochem Biophys Res Commun, 2003. 309(1): p. 232-40.
42. Kim, K.J., et al., Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature, 1993. 362(6423): p. 841-4.
43. Coussens, L.M. and Z. Werb, Inflammation and cancer. Nature, 2002. 420(6917): p. 860-7.
44. de Visser, K.E., L.V. Korets, and L.M. Coussens, De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent. Cancer Cell, 2005. 7(5): p. 411-23.
45. Kalluri, R. and M. Zeisberg, Fibroblasts in cancer. Nat Rev Cancer, 2006. 6(5): p. 392-401.
46. Mueller, M.M. and N.E. Fusenig, Friends or foes - bipolar effects of the tumour stroma in cancer. Nat Rev Cancer, 2004. 4(11): p. 839-49.
47. Ronnov-Jessen, L., O.W. Petersen, and M.J. Bissell, Cellular changes involved in conversion of normal to malignant breast: importance of the stromal reaction. Physiol Rev, 1996. 76(1): p. 69-125.
48. Lazard, D., et al., Expression of smooth muscle-specific proteins in myoepithelium and stromal myofibroblasts of normal and malignant human breast tissue. Proc Natl Acad Sci U S A, 1993. 90(3): p. 999-1003.
49. Schurch, W., T.A. Seemayer, and G. Gabbiani, The myofibroblast: a quarter century after its discovery. Am J Surg Pathol, 1998. 22(2): p. 141-7.
50. Camps, J.L., et al., Fibroblast-mediated acceleration of human epithelial tumor growth in vivo. Proc Natl Acad Sci U S A, 1990. 87(1): p. 75-9.
51. Gleave, M., et al., Acceleration of human prostate cancer growth in vivo by factors produced by prostate and bone fibroblasts. Cancer Res, 1991. 51(14): p. 3753-61.
52. Olumi, A.F., et al., Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res, 1999. 59(19): p. 5002-11.
53. Tlsty, T.D. and P.W. Hein, Know thy neighbor: stromal cells can contribute oncogenic signals. Curr Opin Genet Dev, 2001. 11(1): p. 54-9.
54. Dong-Le Bourhis, X., et al., Effect of stromal and epithelial cells derived from normal and tumorous breast tissue on the proliferation of human breast cancer cell lines in co-culture. Int J Cancer, 1997. 71(1): p. 42-8.
55. Brouty-Boye, D., et al., Fibroblast-mediated differentiation in human breast carcinoma cells (MCF-7) grown as nodules in vitro. Int J Cancer, 1994. 56(5): p. 731-5.
56. Cheng, J.D., et al., Promotion of tumor growth by murine fibroblast activation protein, a serine protease, in an animal model. Cancer Res, 2002. 62(16): p. 4767-72.
57. Loeffler, M., et al., Targeting tumor-associated fibroblasts improves cancer chemotherapy by increasing intratumoral drug uptake. J Clin Invest, 2006. 116(7): p. 1955-62.
58. Apte, M.V., et al., Periacinar stellate shaped cells in rat pancreas: identification, isolation, and culture. Gut, 1998. 43(1): p. 128-33.
59. Geerts, A., History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells. Semin Liver Dis, 2001. 21(3): p. 311-35.
60. Bachem, M.G., et al., Identification, culture, and characterization of pancreatic stellate cells in rats and humans. Gastroenterology, 1998. 115(2): p. 421-32.
61. Omary, M.B., et al., The pancreatic stellate cell: a star on the rise in pancreatic diseases. J Clin Invest, 2007. 117(1): p. 50-9.
62. Haber, P.S., et al., Activation of pancreatic stellate cells in human and experimental pancreatic fibrosis. Am J Pathol, 1999. 155(4): p. 1087-95.
63. Zimmermann, A., et al., Pancreatic stellate cells contribute to regeneration early after acute necrotising pancreatitis in humans. Gut, 2002. 51(4): p. 574-8.
64. Hartel, M., et al., Desmoplastic reaction influences pancreatic cancer growth behavior. World J Surg, 2004. 28(8): p. 818-25.
65. Watanabe, I., et al., Advanced pancreatic ductal cancer: fibrotic focus and beta-catenin expression correlate with outcome. Pancreas, 2003. 26(4): p. 326-33.
66. Vaquero, E.C., et al., Extracellular matrix proteins protect pancreatic cancer cells from death via mitochondrial and nonmitochondrial pathways. Gastroenterology, 2003. 125(4): p. 1188-202.
67. Munshi, H.G. and M.S. Stack, Reciprocal interactions between adhesion receptor signaling and MMP regulation. Cancer Metastasis Rev, 2006. 25(1): p. 45-56.
68. Yamamoto, H., et al., Relation of enhanced secretion of active matrix metalloproteinases with tumor spread in human hepatocellular carcinoma. Gastroenterology, 1997. 112(4): p. 1290-6.
69. Bhatia, S.N., et al., Effect of cell-cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells. FASEB J, 1999. 13(14): p. 1883-900.
70. Grinnell, A.D., Dynamics of nerve-muscle interaction in developing and mature neuromuscular junctions. Physiol Rev, 1995. 75(4): p. 789-834.
71. Shekhar, M.P., et al., Breast stroma plays a dominant regulatory role in breast epithelial growth and differentiation: implications for tumor development and progression. Cancer Res, 2001. 61(4): p. 1320-6.
72. Streuli, C., Extracellular matrix remodelling and cellular differentiation. Curr Opin Cell Biol, 1999. 11(5): p. 634-40.
73. Aboseif, S., et al., Mesenchymal reprogramming of adult human epithelial differentiation. Differentiation, 1999. 65(2): p. 113-8.
74. Frisch, S.M. and E. Ruoslahti, Integrins and anoikis. Curr Opin Cell Biol, 1997. 9(5): p. 701-6.
75. Zhang, H.Z., et al., Estrogen mediates mammary epithelial cell proliferation in serum-free culture indirectly via mammary stroma-derived hepatocyte growth factor. Endocrinology, 2002. 143(9): p. 3427-34.
76. Chang, H.Y., et al., Gene expression signature of fibroblast serum response predicts human cancer progression: similarities between tumors and wounds. PLoS Biol, 2004. 2(2): p. E7.
77. Kuperwasser, C., et al., Reconstruction of functionally normal and malignant human breast tissues in mice. Proc Natl Acad Sci U S A, 2004. 101(14): p. 4966-71.
78. Vonlaufen, A., et al., Pancreatic stellate cells: partners in crime with pancreatic cancer cells. Cancer Res, 2008. 68(7): p. 2085-93.
79. Friedman, S.L., Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies. N Engl J Med, 1993. 328(25): p. 1828-35.
80. Schmitt, C.A., et al., A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy. Cell, 2002. 109(3): p. 335-46.
81. Ly, D.H., et al., Mitotic misregulation and human aging. Science, 2000. 287(5462): p. 2486-92.
82. Toussaint, O., et al., From the Hayflick mosaic to the mosaics of ageing. Role of stress-induced premature senescence in human ageing. Int J Biochem Cell Biol, 2002. 34(11): p. 1415-29.
83. Toussaint, O., E.E. Medrano, and T. von Zglinicki, Cellular and molecular mechanisms of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes. Exp Gerontol, 2000. 35(8): p. 927-45.
84. te Poele, R.H., et al., DNA damage is able to induce senescence in tumor cells in vitro and in vivo. Cancer Res, 2002. 62(6): p. 1876-83.
85. Chang, B.D., et al., Molecular determinants of terminal growth arrest induced in tumor cells by a chemotherapeutic agent. Proc Natl Acad Sci U S A, 2002. 99(1): p. 389-94.
86. Chang, B.D., et al., A senescence-like phenotype distinguishes tumor cells that undergo terminal proliferation arrest after exposure to anticancer agents. Cancer Res, 1999. 59(15): p. 3761-7.
87. Millis, A.J., et al., Differential expression of metalloproteinase and tissue inhibitor of metalloproteinase genes in aged human fibroblasts. Exp Cell Res, 1992. 201(2): p. 373-9.
88. Shelton, D.N., et al., Microarray analysis of replicative senescence. Curr Biol, 1999. 9(17): p. 939-45.
89. West, M.D., O.M. Pereira-Smith, and J.R. Smith, Replicative senescence of human skin fibroblasts correlates with a loss of regulation and overexpression of collagenase activity. Exp Cell Res, 1989. 184(1): p. 138-47.
90. Wick, M., et al., A novel member of human tissue inhibitor of metalloproteinases (TIMP) gene family is regulated during G1 progression, mitogenic stimulation, differentiation, and senescence. J Biol Chem, 1994. 269(29): p. 18953-60.
91. Schwarze, S.R., et al., Novel pathways associated with bypassing cellular senescence in human prostate epithelial cells. J Biol Chem, 2002. 277(17): p. 14877-83.
92. Kirkwood, T.B. and S.N. Austad, Why do we age? Nature, 2000. 408(6809): p. 233-8.
93. Krtolica, A., et al., Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging. Proc Natl Acad Sci U S A, 2001. 98(21): p. 12072-7.
94. Chang, B.D., et al., Effects of p21Waf1/Cip1/Sdi1 on cellular gene expression: implications for carcinogenesis, senescence, and age-related diseases. Proc Natl Acad Sci U S A, 2000. 97(8): p. 4291-6.
95. Tsai, K.K., et al., Cellular mechanisms for low-dose ionizing radiation-induced perturbation of the breast tissue microenvironment. Cancer Res, 2005. 65(15): p. 6734-44.
96. Liu, D. and P.J. Hornsby, Senescent human fibroblasts increase the early growth of xenograft tumors via matrix metalloproteinase secretion. Cancer Res, 2007. 67(7): p. 3117-26.
97. Jesnowski, R., et al., Immortalization of pancreatic stellate cells as an in vitro model of pancreatic fibrosis: deactivation is induced by matrigel and N-acetylcysteine. Lab Invest, 2005. 85(10): p. 1276-91.
98. Dimri, G.P., et al., A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A, 1995. 92(20): p. 9363-7.
99. Kimball, R.E., et al., Flow cytometric analysis of lymph node metastases in advanced ovarian cancer: clinical and biologic significance. Am J Obstet Gynecol, 1997. 176(6): p. 1319-26; discussion 1326-7.
100. Wang, X. and B. Seed, A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Res, 2003. 31(24): p. e154.
101. Mogal, A. and S.A. Abdulkadir, Effects of Histone Deacetylase Inhibitor (HDACi); Trichostatin-A (TSA) on the expression of housekeeping genes. Mol Cell Probes, 2006. 20(2): p. 81-6.
102. Lee, G.Y., et al., Three-dimensional culture models of normal and malignant breast epithelial cells. Nat Methods, 2007. 4(4): p. 359-65.
103. Hoffman, R.M. and M. Yang, Subcellular imaging in the live mouse. Nat Protoc, 2006. 1(2): p. 775-82.
104. Bouvet, M., et al., In vivo color-coded imaging of the interaction of colon cancer cells and splenocytes in the formation of liver metastases. Cancer Res, 2006. 66(23): p. 11293-7.
105. Niggli, H.J., et al., Mitomycin C-induced postmitotic fibroblasts retain the capacity to repair pyrimidine photodimers formed after UV-irradiation. Mutat Res, 1989. 219(4): p. 231-40.
106. Rodemann, H.P., et al., Selective enrichment and biochemical characterization of seven human skin fibroblasts cell types in vitro. Exp Cell Res, 1989. 180(1): p. 84-93.
107. van Moorsel, C.J., G.J. Peters, and H.M. Pinedo, Gemcitabine: Future Prospects of Single-Agent and Combination Studies. Oncologist, 1997. 2(3): p. 127-134.
108. Veltkamp, S.A., J.H. Beijnen, and J.H. Schellens, Prolonged versus standard gemcitabine infusion: translation of molecular pharmacology to new treatment strategy. Oncologist, 2008. 13(3): p. 261-76.
109. Horan, P.K. and S.E. Slezak, Stable cell membrane labelling. Nature, 1989. 340(6229): p. 167-8.
110. Majeti, R., et al., Dysregulated gene expression networks in human acute myelogenous leukemia stem cells. Proc Natl Acad Sci U S A, 2009. 106(9): p. 3396-401.
111. Jemal, A., et al., Cancer statistics, 2009. CA Cancer J Clin, 2009. 59(4): p. 225-49.
112. Bachem, M.G., et al., Pancreatic carcinoma cells induce fibrosis by stimulating proliferation and matrix synthesis of stellate cells. Gastroenterology, 2005. 128(4): p. 907-21.
113. Thiery, J.P., Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer, 2002. 2(6): p. 442-54.
114. Yang, A.D., et al., Chronic oxaliplatin resistance induces epithelial-to-mesenchymal transition in colorectal cancer cell lines. Clin Cancer Res, 2006. 12(14 Pt 1): p. 4147-53.
115. Hiscox, S., et al., Tamoxifen resistance in breast cancer cells is accompanied by an enhanced motile and invasive phenotype: inhibition by gefitinib ('Iressa', ZD1839). Clin Exp Metastasis, 2004. 21(3): p. 201-12.
116. Hiscox, S., et al., Tamoxifen resistance in MCF7 cells promotes EMT-like behaviour and involves modulation of beta-catenin phosphorylation. Int J Cancer, 2006. 118(2): p. 290-301.
117. Li, H., X. Fan, and J. Houghton, Tumor microenvironment: the role of the tumor stroma in cancer. J Cell Biochem, 2007. 101(4): p. 805-15.
118. Kurz, D.J., et al., Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells. J Cell Sci, 2000. 113 ( Pt 20): p. 3613-22.
119. Hayflick, L. and P.S. Moorhead, The serial cultivation of human diploid cell strains. Exp Cell Res, 1961. 25: p. 585-621.
120. Roninson, I.B., Tumor cell senescence in cancer treatment. Cancer Res, 2003. 63(11): p. 2705-15.
121. Lanz, C., et al., Rapid determination of gemcitabine in plasma and serum using reversed-phase HPLC. J Sep Sci, 2007. 30(12): p. 1811-20.
122. Fakih, M.G., et al., Phase I and pharmacokinetic study of weekly docetaxel, cisplatin, and daily capecitabine in patients with advanced solid tumors. Clin Cancer Res, 2005. 11(16): p. 5942-9.
123. Massova, I., et al., Matrix metalloproteinases: structures, evolution, and diversification. FASEB J, 1998. 12(12): p. 1075-95.
124. Stamenkovic, I., Matrix metalloproteinases in tumor invasion and metastasis. Semin Cancer Biol, 2000. 10(6): p. 415-33.
125. Walker, R.A., The complexities of breast cancer desmoplasia. Breast Cancer Res, 2001. 3(3): p. 143-5.
126. Burris, H., 3rd and C. Rocha-Lima, New therapeutic directions for advanced pancreatic cancer: targeting the epidermal growth factor and vascular endothelial growth factor pathways. Oncologist, 2008. 13(3): p. 289-98.
127. Wilson, J. and F. Balkwill, The role of cytokines in the epithelial cancer microenvironment. Semin Cancer Biol, 2002. 12(2): p. 113-20.
128. Arenberg, D.A., et al., Epithelial-neutrophil activating peptide (ENA-78) is an important angiogenic factor in non-small cell lung cancer. J Clin Invest, 1998. 102(3): p. 465-72.
129. Yoneda, J., et al., Expression of angiogenesis-related genes and progression of human ovarian carcinomas in nude mice. J Natl Cancer Inst, 1998. 90(6): p. 447-54.
130. Luan, J., et al., Mechanism and biological significance of constitutive expression of MGSA/GRO chemokines in malignant melanoma tumor progression. J Leukoc Biol, 1997. 62(5): p. 588-97.
131. Takamori, H., et al., Autocrine growth effect of IL-8 and GROalpha on a human pancreatic cancer cell line, Capan-1. Pancreas, 2000. 21(1): p. 52-6.
132. Koshiba, T., et al., Expression of stromal cell-derived factor 1 and CXCR4 ligand receptor system in pancreatic cancer: a possible role for tumor progression. Clin Cancer Res, 2000. 6(9): p. 3530-5.
133. Orimo, A., et al., Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell, 2005. 121(3): p. 335-48.
134. Lev, D.C., et al., Exposure of melanoma cells to dacarbazine results in enhanced tumor growth and metastasis in vivo. J Clin Oncol, 2004. 22(11): p. 2092-100.
135. Olnes, M.J. and R. Erlich, A review and update on cholangiocarcinoma. Oncology, 2004. 66(3): p. 167-79.
136. Shaib, Y. and H.B. El-Serag, The epidemiology of cholangiocarcinoma. Semin Liver Dis, 2004. 24(2): p. 115-25.
137. Klempnauer, J., et al., What constitutes long-term survival after surgery for hilar cholangiocarcinoma? Cancer, 1997. 79(1): p. 26-34.
138. Burke, E.C., et al., Hilar Cholangiocarcinoma: patterns of spread, the importance of hepatic resection for curative operation, and a presurgical clinical staging system. Ann Surg, 1998. 228(3): p. 385-94.
139. Alberts, S.R., et al., Treatment options for hepatobiliary and pancreatic cancer. Mayo Clin Proc, 2007. 82(5): p. 628-37.
140. Maruyama, M., et al., Establishment of a highly differentiated immortalized human cholangiocyte cell line with SV40T and hTERT. Transplantation, 2004. 77(3): p. 446-51.

------------------------------------------------------------------------ 第 12 筆 ---------------------------------------------------------------------
系統識別號 U0007-2407200911053400
論文名稱(中文) 低鈣狀態對於發育中大白鼠神經系統BDNF轉錄之影響
論文名稱(英文) The effect of hypocalcemia on the transcription of BDNF in the developing central nervous system of rat
校院名稱 臺北醫學大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Graduate Institute of Clinical Medicine
學年度 97
學期 2
出版年 98
研究生(中文) 李淑婷
學號 M118096006
學位類別 碩士
語文別 中文
口試日期 2009-07-07
論文頁數 54頁
口試委員 指導教授-葉健全
共同指導教授-洪焜隆
委員-陳景宗
委員-李怡萱
委員-沈芯?{
關鍵字(中) 低血鈣
神經發育
關鍵字(英) hypocalcemia
BDNF
neurodevelopment
學科別分類
中文摘要 新生兒低血鈣是臨床上常見的現象,然而新生兒期暫時性低血鈣是否會造成未來神經發展的異常,鮮少有人探討。本實驗室的前趨研究發現,低鈣之培養液會造成大白鼠胚胎神經細胞明顯的死亡,鈣離子濃度越低,細胞死亡的情形越嚴重。表示低血鈣對於不成熟的神經系統可能具有神經毒性。
神經活動依賴之可塑性(activity-dependent neuronal plasticity) 亦即神經細胞的活動會造成本身及鄰近細胞功能及型態的長期性改變是神經細胞發展過程最重要的一環。近來的研究顯示,neuronal plasticity的形成與神經營養因子(neurotrophin)蛋白質有極密切的關係。其中之一便是BDNF(Brain-derived neurotrophic factor)。BDNF的基因含有8個5’端不轉譯的exon(exonI~VIII)及一個3’端的蛋白轉譯exon (exon IX)。BDNF的mRNA transcript一共有22種,exon IX BDNF mRNA為各transcript所共有,也是後來真正轉譯成蛋白質的部份,它的表現會隨著神經細胞的活動而增加,並且這種增加必須伴隨著細胞內鈣離子濃度的上升。Exon I-IX BDNF(BDNF-I) mRNA則為腦部所特有,在腦外組織表現量極少。過去研究發現,從不同鈣離子通道進入的鈣離子會活化不同的exon primer,而作出不同的BDNF mRNA transcript。因此,神經細胞外鈣離子降低有可能改變BDNF的表現量。為測試此假設我們使用大白鼠胚胎的大腦皮質細胞,作初級培養,並給予持續低鈣或暫時低鈣處理,觀察神經纖維的生長情形及total BDNF mRNA(exonIX)以及BDNF-I mRNA的表現量是否有所不同並且偵測其神經纖維生長狀況及粗細(活體外實驗)。另外我們也以短期產前皮下注射calcitonin導致初生幼鼠有短暫血鈣下降的動物模式,觀察出生後生長狀況,並偵測其海馬迴total BDNF mRNA及BDNF-I mRNA 表現量的變化。細胞模式實驗發現持續低鈣組在6天時,輕微低鈣(1.75mM)會使total BDNF mRNA下降,但嚴重低鈣(0.96m,0.31mM)反而會上升;BDNF-I mRNA則會下降。暫時低鈣組在12天時的total BDNF mRNA及BDNF-I mRNA均下降。持續低鈣0.31mM組在培養3天後神經纖維生長明顯比正常組短;培養到12天後,0.96m及0.31mM組的神經纖維直徑明顯較對照組細胞細。另一方面,我們將懷孕母鼠在產前連續二天給予calcitonin皮下注射使其產生血鈣降低(活體內實驗),其所生幼鼠於出生一天時有短暫性低血鈣,且發現低鈣母鼠所生之幼鼠在出生後7天內的身長體重明顯低於對照組。此外,低鈣組幼鼠其海馬迴之total BDNF mRNA表現量在出生後十四天時有下降的趨勢。
以上結果顯示細胞外鈣離子降低的確會影響BDNF轉譯以及神經纖維的生長,但是二者的變化是否有相關則有待未來繼續研究。
英文摘要 Hypocalcaemia is a common problem, yet the long-term sequels of transient hypocalcaemia in developing brain are not known and have not been carefully characterized. Our preliminary study revealed that reduced calcium concentration in the culture medium would lead to rat embryonic neuron death, and the proportion of neuron death is related to calcium concentration. These results suggest that hypocalcemia may be neurotoxic for immature neuron.
“Activity-dependent neuronal plasticity” is an important phenomenon during neuron development. One attractive molecular candidates for modulating synaptic plasticity are the member of neurotrophins (NTs) family, include BDNF(Brain-derived neurotrophic factor). BDNF gene includes eight 5’ noncoding exons (exonI~VIII) and one 3’ protein coding exon (exon IX).Rat BDNF mRNA contains 22 transcripts, and exon IX mRNA is common region for all transcripts, which could be up-regulated when neuron activity increase. Furthermore, the up-regulation should accompany with increased intracellular calcium concentration. Exon I-IX BDNF(BDNF-I) mRNA transcript is brain-specific. Extracellular calcium could enter into cell via different calcium channel, which has been shown to activate different exon primer and then induces transcription of BDNF mRNA. We proposed that reduction of extracellular calcilum level might alter the expression of BDNF in transcriptional level. To test this hypothesis, we determined the level of total BDNF and BDNF-I mRNA using Real-Time PCR in primary rat cortical neuron cell culture in present of low calcium medium. In addition, we also determined the BDNF transcript in the hippocampus of developing rats born to dam rats received 2-day injection of calcitonin before delivery.
Long-term incubation of cells in low calcium medium induces a transient decrease or increase in total BDNF mRNA and BDNF-1 mRNA, which dependent on the concentration of calcium and incubation period. On the other hand, transient low calcium incubation on Day in vitro 1(DIV 1) induced a delay-onset of reduction of total BDNF and BDNF-I mRNA determined on DV12. Incubation in very low extracellular calcium (0.31mM) medium induced a reduction in the neurite growth and the diameter. Rats born to calcitonin-injected dams rats had low serum calcium level, low body weight gain, and body length growth during the first postnatal week. In addition, total BDNF mRNA level has significant trend of decrease on post-natal day 14.
This result demonstrated that lower extracellular calcium level could affect the transcription of BDNF and neuronal growth. However, whether the reduction of BDNF expression is related to the abnormal neurite growth is required further investigation.
論文目次 中文摘要 IV
英文摘要 VI
第壹章 緒論 1
壹、低血鈣在臨床上的影響 2
貳、鈣與神經發育的關係 3
參、BDNF與神經發育的相關性 6
肆、實驗計劃目的 11
第貳章 實驗材料及方法 13
壹、實驗材料 14
貳、實驗方法 17
第參章 實驗結果 23
壹、初級培養之胚胎大腦皮質神經細胞低鈣培養下神經纖維的生長狀況 24
貳、初級培養之胚胎大腦皮質神經細胞低鈣培養下total BDNF mRNA及 BDNF-I mRNA的表現量 25
參、低鈣組與控制組之新生幼鼠的生長情況比較 26
肆、低鈣之新生幼鼠腦部的total BDNF mRNA及I BDNF-I mRNA的表現量 26
第肆章 實驗討論 27
參考文獻 35
圖表 41
參考文獻 Aid T, Kazantseva A, Piirsoo M, Palm K, Timmusk T. (2007). "Mouse and rat BDNF gene structure and expression revisited." J Neurosci Res 85(3): 525-35.
Bading H, Ginty DD, Greenberg ME. (1993). "Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways." Science 260(5105): 181-6.
Balasubramanian S, Shivbalan S, Kumar PS. (2006). "Hypocalcemia due to vitamin D deficiency in exclusively breastfed infants." Indian Pediatr 43(3): 247-51.
Bhowmick SK. (2007). "A neonate with persistent twitching. Diagnosis: neonatal hypocalcemia." Clin Pediatr (Phila) 46(5): 459-61.
Bixby JL, Spitzer NC. (1984). "Early differentiation of vertebrate spinal neurons in the absence of voltage-dependent Ca2+ and Na+ influx." Dev Biol 106(1): 89-96.
Castrén E, Zafra F, Thoenen H, Lindholm D. (1992). "Light regulates expression of brain-derived neurotrophic factor mRNA in rat visual cortex." Proc Natl Acad Sci U S A 89(20): 9444-8.
Chen WG, West AE, Tao X, Corfas G, Szentirmay MN, Sawadogo M, Vinson C, Greenberg ME. (2003). "Upstream stimulatory factors are mediators of Ca2+-responsive transcription in neurons." J Neurosci 23(7): 2572-81.
Cockburn F, Brown JK, Belton NR, Forfar JO. (1973). "Neonatal convulsions associated with primary disturbance of calcium, phosphorus, and magnesium metabolism." Arch Dis Child 48(2): 99-108.
Cormier RJ, Greenwood AC, Connor JA. (2001). "Bidirectional synaptic plasticity correlated with the magnitude of dendritic calcium transients above a threshold." J Neurophysiol 85(1): 399-406.
Ernfors P, Lee KF, Jaenisch R. (1994). "Mice lacking brain-derived neurotrophic factor develop with sensory deficits." Nature 368(6467): 147-50.
Ernfors P, Wetmore C, Olson L, Persson H. (1990). "Identification of cells in rat brain and peripheral tissues expressing mRNA for members of the nerve growth factor family." Neuron 5(4): 511-26.
Figurov A, Pozzo-Miller LD, Olafsson P, Wang T, Lu B. (1996). "Regulation of synaptic responses to high-frequency stimulation and LTP by neurotrophins in the hippocampus." Nature 381(6584): 706-9.
Friedman WJ, Olson L, Persson H. (1991). "Cells that Express Brain-Derived Neurotrophic Factor mRNA in the Developing Postnatal Rat Brain." Eur J Neurosci 3(7): 688-697.
Ghosh A, Carnahan J, Greenberg ME. (1994). "Requirement for BDNF in activity-dependent survival of cortical neurons." Science 263(5153): 1618-23.
Gu X, Spitzer NC. (1995). "Distinct aspects of neuronal differentiation encoded by frequency of spontaneous Ca2+ transients." Nature 375(6534): 784-7.
Hansel C, Artola A, Singer W. (1997). "Relation between dendritic Ca2+ levels and the polarity of synaptic long-term modifications in rat visual cortex neurons." Eur J Neurosci 9(11): 2309-22.
Hebb.DO (1949). "The organization of behavior."
Holliday J, Spitzer NC. (1990). "Spontaneous calcium influx and its roles in differentiation of spinal neurons in culture." Dev Biol 141(1): 13-23.
Holliday J, Spitzer NC. (1993). "Calcium regulates neuronal differentiation both directly and via co-cultured myocytes." J Neurobiol 24(4): 506-14.
Jones KR, Fariñas I, Backus C, Reichardt LF. (1994). "Targeted disruption of the BDNF gene perturbs brain and sensory neuron development but not motor neuron development." Cell 76(6): 989-99.
Keen JH. (1969). "Significance of hypocalcaemia in neonatal convulsions." Arch Dis Child 44(235): 356-61.
Kingsbury TJ, Murray PD, Bambrick LL, Krueger BK. (2003). "Ca(2+)-dependent regulation of TrkB expression in neurons." J Biol Chem 278(42): 40744-8.
Korte M, Carroll P, Wolf E, Brem G, Thoenen H, Bonhoeffer T. (1995). "Hippocampal long-term potentiation is impaired in mice lacking brain-derived neurotrophic factor." Proc Natl Acad Sci U S A 92(19): 8856-60.
Lisman J. (1989). "A mechanism for the Hebb and the anti-Hebb processes underlying learning and memory." Proc Natl Acad Sci U S A 86(23): 9574-8.
Liu QR, Walther D, Drgon T, Polesskaya O, Lesnick TG, Strain KJ, de Andrade M, Bower JH, Maraganore DM, Uhl GR. (2005). "Human brain derived neurotrophic factor (BDNF) genes, splicing patterns, and assessments of associations with substance abuse and Parkinson's Disease." Am J Med Genet B Neuropsychiatr Genet 134B(1): 93-103.
Lynch BJ, Rust RS. (1994). "Natural history and outcome of neonatal hypocalcemic and hypomagnesemic seizures." Pediatr Neurol 11(1): 23-7.
Magee JC, Johnston D. (1997). "A synaptically controlled, associative signal for Hebbian plasticity in hippocampal neurons." Science 275(5297): 209-13.
McInerny TK, Schubert WK. (1969). "Prognosis of neonatal seizures." Am J Dis Child 117(3): 261-4.
Murphy TH, Worley PF, Baraban JM. (1991). "L-type voltage-sensitive calcium channels mediate synaptic activation of immediate early genes." Neuron 7(4): 625-35.
Neveu D, Zucker RS. (1996). "Postsynaptic levels of [Ca2+]i needed to trigger LTD and LTP." Neuron 16(3): 619-29.
Nishiyama M, Hong K, Mikoshiba K, Poo MM, Kato K. (2000). "Calcium stores regulate the polarity and input specificity of synaptic modification." Nature 408(6812): 584-8.
Patterson SL, Abel T, Deuel TA, Martin KC, Rose JC, Kandel ER. (1996). "Recombinant BDNF rescues deficits in basal synaptic transmission and hippocampal LTP in BDNF knockout mice." Neuron 16(6): 1137-45.
Pozzo-Miller LD, Gottschalk W, Zhang L, McDermott K, Du J, Gopalakrishnan R, Oho C, Sheng ZH, Lu B. (1999). "Impairments in high-frequency transmission, synaptic vesicle docking, and synaptic protein distribution in the hippocampus of BDNF knockout mice." J Neurosci 19(12): 4972-83.
Rose AL, Lombroso CT. (1970). "A study of clinical, pathological, and electroencephalographic features in 137 full-term babies with a long-term follow-up." Pediatrics 45(3): 404-25.
Sabatini BL, Maravall M, Svoboda K. (2001). "Ca(2+) signaling in dendritic spines." Curr Opin Neurobiol 11(3): 349-56.
Sanna B, Kramer D, Genazzani AA. (2002). "The expression of the PDZ protein MALS-1/velis is regulated by calcium and calcineurin in cerebellar granule cells." J Biol Chem 277(51): 49585-90.
Schiller J, Schiller Y, Clapham DE. (1998). "NMDA receptors amplify calcium influx into dendritic spines during associative pre- and postsynaptic activation." Nat Neurosci 1(2): 114-8.
Sheth DP. (1997). "Hypocalcemic seizures in neonates." Am J Emerg Med 15(7): 638-41.
Shieh PB, Hu SC, Bobb K, Timmusk T, Ghosh A. (1998). "Identification of a signaling pathway involved in calcium regulation of BDNF expression." Neuron 20(4): 727-40.
Spitzer NC. (2002). "Activity-dependent neuronal differentiation prior to synapse formation: the functions of calcium transients." J Physiol Paris 96(1-2): 73-80.
Spoerri PE, Dozier AK, Roisen FJ. (1990). "Calcium regulation of neuronal differentiation: the role of calcium in GM1-mediated neuritogenesis." Brain Res Dev Brain Res 56(2): 177-88.
Tabuchi A. (2008). "Synaptic plasticity-regulated gene expression: a key event in the long-lasting changes of neuronal function." Biol Pharm Bull 31(3): 327-35.
Tabuchi A, Nakaoka R, Amano K, Yukimine M, Andoh T, Kuraishi Y, Tsuda M. (2000). "Differential activation of brain-derived neurotrophic factor gene promoters I and III by Ca2+ signals evoked via L-type voltage-dependent and N-methyl-D-aspartate receptor Ca2+ channels." J Biol Chem 275(23): 17269-75.
Tabuchi A, Sakaya H, Kisukeda T, Fushiki H, Tsuda M. (2002). "Involvement of an upstream stimulatory factor as well as cAMP-responsive element-binding protein in the activation of brain-derived neurotrophic factor gene promoter I." J Biol Chem 277(39): 35920-31.
Tao X, Finkbeiner S, Arnold DB, Shaywitz AJ, Greenberg ME. (1998). "Ca2+ influx regulates BDNF transcription by a CREB family transcription factor-dependent mechanism." Neuron 20(4): 709-26.
Tao X, West AE, Chen WG, Corfas G, Greenberg ME. "A calcium-responsive transcription factor, CaRF, that regulates neuronal activity-dependent expression of BDNF." Neuron 33(3): 383-95.
Thoenen H. (1995). "Neurotrophins and neuronal plasticity." Science 270(5236): 593-8.
Timmusk T, Palm K, Metsis M, Reintam T, Paalme V, Saarma M, Persson H. (1993). "Multiple promoters direct tissue-specific expression of the rat BDNF gene." Neuron 10(3): 475-89.
Yan Q, Rosenfeld RD, Matheson CR, Hawkins N, Lopez OT, Bennett L, Welcher AA. (1997). "Expression of brain-derived neurotrophic factor protein in the adult rat central nervous system." Neuroscience 78(2): 431-48.
Yang SN, Tang YG, Zucker RS. (1999). "Selective induction of LTP and LTD by postsynaptic [Ca2+]i elevation." J Neurophysiol 81(2): 781-7.

------------------------------------------------------------------------ 第 13 筆 ---------------------------------------------------------------------
系統識別號 U0007-3001201017520100
論文名稱(中文) 回復尋常飲食比低劑量銀杏葉萃取物對大白鼠非酒精性脂肪肝炎的進展更具有抑制效果
論文名稱(英文) Return to ordinary diet has a better inhibition than low dose of Ginkgo biloba extract on the progression ofnon-alcoholic steatohepatitis in rat
校院名稱 臺北醫學大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Graduate Institute of Clinical Medicine
學年度 98
學期 1
出版年 99
研究生(中文) 陳 永 發
學號 M102095032
學位類別 碩士
語文別 中文
口試日期 2009-12-26
論文頁數 91頁
口試委員 指導教授-陳盛煊
委員-楊賢馨
委員-陳俊榮
關鍵字(中) 非酒精性脂肪肝炎
銀杏葉萃取物
氧化壓力
動物模式
關鍵字(英) non-alcoholic steatohepatitis
Ginkgo biloba extract
oxidative stress
animal model
學科別分類
中文摘要 非酒精性脂肪肝病(non-alcoholic fatty liver disease, NAFLD)在西方國家盛行率大約10 ~24%,其中約2~3%是以慢性肝炎為表現,文獻指出在5~13年的追蹤中,約三分之一的病患,其肝臟纖維化程度會加劇,最終可能會演變成肝硬化或產生肝癌,目前對於非酒精性脂肪肝炎(non-alcoholic steatohepatitis, NASH)的治療,除減重、運動來減少胰島素抗性外,並無特定療效的藥物。銀杏葉萃取物是保健的中草藥,在以往使用上並無明顯或重大的副作用,近年來發現它具有很強的清除自由基能力及抗氧化能力,所以本研究目的在於觀察銀杏葉萃取物是否對大白鼠非酒精性脂肪肝炎的進展具有預防效果。在餵食雄性Wistar大白鼠十六週液態高油脂飲食,經病理切片證實誘發NASH後,隨機分為三組:繼續液態高油飲食組(H組)、繼續液態高油飲食加銀杏葉萃取物組(HG組)及恢復尋常固型飼料組(N組),再飼養六週後犧牲,銀杏葉萃取物是採腹腔內注射,劑量為每天每公斤體重25毫克。結果發現在生化檢驗方面,HG組與H組相比只有血清三酸甘油酯及血糖為有意義的下降(P<0.05),其餘如肝功能指數、總膽固醇和血清胰島素均無差別。在肝臟生化檢驗方面,除了游離脂肪酸(free fatty acid, FFA)為有意義的下降外(P<0.01),glutathione reductase、 glutathione peroxidase(GPx)、 superoxide dismutase(SOD)、 lipid peroxidation、myeloperoxidase及tumor necrosis factor-α (TNF-α)也均無差別。另外血清TNF-α、hyaluronic acid及FFA雖有下降的趨勢,但其差別無達到統計上的意義。最後病理組織結果顯示所有HG組其NAS ( NAFLD activity score )都大於或等於五分,持續NASH的病理診斷。相反的,所有N組其NAS都小於五分,病理組織顯示脂肪堆積(P<0.01)及小葉發炎(P<0.05)與H組相比具有統計意義的下降,其次血糖、血清胰島素、肝臟FFA及血清TNF-α的下降和肝臟GPx、SOD的上昇與H組相比,亦具有統計上的差別(P<0.05)。由研究結果顯示補充六週低劑量的銀杏葉萃取物,無法明顯地減少大白鼠的氧化壓力,連帶著也無法防止大白鼠NASH的持續進展,不過經由減少飲食內脂肪的比例,來達到總熱量攝取的減少,是可以逆轉大白鼠NASH的進展。所以是否改變藥物的投予方式(例如改為口服)或延長給藥期間來增加總劑量,而達到實驗成功,是值得我們再去探討的一個議題,另外經由這實驗也證實了飲食、熱量的控制是改善NASH的首要且有效的措施。
英文摘要 The prevalence of non-alcoholic fatty liver disease (NAFLD) is about 10~24% in the West. Approximately 2~3% of the presentations about this disease were chronic hepatitis and eventually liver cirrhosis or hepatocellular carcinoma may develop. Because lack of specific efficacy of drugs except weight loss, exercise and improvement of insulin resistance, non-alcoholic steatohepatitis (NASH) will surely become an important issue in the future. Strong free radical scavenging ability and antioxidant capacity of Ginkgo biloba extract(EGb 761)has been found in recent years, so the purpose of this study is to observe whether the EGb 761 has the preventive effect on the progression of NASH. NASH confirmed by liver pathology after liquid high-fat diet feeding 16 weeks, these male Wistar rats were randomly divided and fed as following 3 groups for more 6 weeks : liquid high-fat diet group (H group), liquid high-fat diet plus EGb 761 group ( HG group) and ordinary solid diet group (N group). EGb761 was delivered by intra-peritoneal injection with the daily dose of 25 mg/ kg of body weight. Only serum triglyceride and glucose significantly decreased in HG group compared with H group in blood biochemistry(P<0.05). Others, including liver function test, total cholesterol and serum insulin had no difference between these 2 groups. Except for a significant decline of hepatic free fatty acid concentration in HG group(P<0.01), the glutathione reductase, glutathione peroxidase, superoxide dismutase, lipid peroxidation, myeloperoxidase and tumor necrosis factor-α had no difference in the liver biochemistry. Although there was a downward trend about the serum concentration of tumor necrosis factor-α, hyaluronic acid and free fatty acid in HG group compared with H group, they still can not reach significant differences. The liver pathologic results showed that the NAS (NAFLD activity score) was more than or equal to five points in all HG group. On the contrary, the NAS of rats in N group were all less than 5 points. The final results showed that the six-week supplement of low dose of EGb 761 can not significantly reduced oxidative stress and prevent the progression of NASH in rats. But it can be achieved by decreasing total calories intake with less fat content in diet. So it is worthy to investigate how to increase the total dose of EGb 761 by changing the route of delivery or extending the duration of experiment. Meanwhile this study proved that restriction of calories intake should be the first task and the most effective in management of NSAH.
論文目次 中文摘要 ……………………………………………… i
英文摘要 ……………………………………………… iii
誌謝 …………………………………………………… iv
目錄 ……………………………………………………… v
圖目錄 …………………………………………………… Vii
表目錄 …………………………………………………… Viii
第一章 緒論
第一節 前言………………………………………… 1
第二節 研究動機與目的 …………………………… 4
第二章 文獻回顧
第一節 非酒精性脂肪肝炎
一、 流行病學上的觀察 ……………………… 6
二、 致病機轉 ………………………………… 8
三、 抗氧化劑的應用 …………………………… 12
第二節 銀杏葉萃取物
一、 生理功能 ………………………………… 16
二、 臨床應用 ………………………………… 18
三、 動物實驗成果 ……………………………… 20
第三章 材料及方法
第一節 實驗動物及特殊材料 ………………………… 27
第二節 實驗設計 ……………………………………… 29
第三節 樣品收集 ……………………………………… 31
第四節 分析項目 ……………………………………… 34
第五節 統計方法 ……………………………………… 47
第四章 結果
第一節 非酒精性脂肪肝炎動物模型誘發實驗
一、 肝臟切片 ……………………………………… 48
二、 體重、肝臟重及肝臟/體重比………………… 48
三、 血液生化值 …………………………………… 49
四、 肝臟生化值 …………………………………… 49
五、 血液細胞激素及游離脂肪酸值 ……………… 50
第二節 銀杏葉萃取物及回復尋常飼料介入實驗
一、 體重、肝臟重及肝臟/體重比 ……………… 57
二、 肝臟切片 ……………………………………… 59
三、 血液生化值 …………………………………… 61
四、 肝臟生化值 …………………………………… 64
五、 血液細胞激素及游離脂肪酸值……………… 67
第五章 討論 ……………………………………………… 69
第六章 結論 ……………………………………………… 81
參考文獻 …………………………………………………… 82
參考文獻 Adachi Y, Moore LE, Bradford BU, Gao W and Thurman RG.(1995)Antibiotics prevent liver injury in rats following long-term exposure to ethanol. Gastroenterology 108: 218-224

Adams LA, Zein CO, Angulo P and Lindor KD (2004) A pilot trial of pentoxifylline in nonalcoholic steatohepatitis. Am J Gastroenterol 99:2365–2368.

Albano E, Mottaran E, Vidali M, Reale E, Saksena S, Occhino G, Burt AD and Day CP (2005) Immune response towards lipid peroxidation products as a predictor of progression of non-alcoholic fatty liver disease to advanced fibrosis. Gut.54: 987-993.

Andersen T and Gluud C (1984) Liver morphology in morbid obesity: a literature study. Int J Obes.8:97-106.

Angulo P (2007) GI epidemiology: nonalcoholic fatty liver disease. Aliment Pharmacol Ther 25 : 883–889

Angulo P and Lindor KD (2002) Non-alcoholic fatty liver disease. J Gastroenterol Hepatol. 17 Suppl:S186-190

Anstee QM and Goldin RD (2006) Mouse models in non-alcoholic fatty liver disease
and steatohepatitis research. Int J Exp Pathol. 87:1-16.

Batman PA and Scheuer PJ (1985) Diabetic hepatitis preceding the onset of glucose intolerance. Histopathology 9:237-243

Bertram SR, Venter I and Stewart RI(1990) Weight loss in obese women—exercise
v. dietary education. S Afr Med J 78:15-18.

Bradford MM ( 1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254

Brevoort P (1998) The booming US botanical market-a new overview. Herbalgram 44: 33-46

Brunt EM, Janney CJ, Di Bisceglie AM, Neuschwander-Tetri BA and Bacon BR (1999)
Nonalcoholic Steatohepatitis: A Proposal for Grading and Staging the Histological Lesions. Am J Gastroenterol. 94:2467-2474

Cai D, Yuan M, Frantz DF, Melendez PA, Hansen L, Lee J and Shoelson SE (2005)
Local and systemic insulin resistance resulting from hepatic activation of IKK-β and NF-κB. Nat Med 11:183-190

Chao CJ, Hung HC, Chen SH and Fang CL (2004) Effects of Ginkgo biloba extract on cytoprotective factors in rats with duodenal ulcer. World J Gastroenterol 10:560-566

Chen SH, Liang YC, Chao JC, Tsai LH, Chang CC, Wang CC amd Pan S (2005) Protective effects of Ginkgo biloba extract on the ethanol-induced gastric ulcer in rats. World J Gastroenterol 11:3746-3750

Cheung F, Siow YL, Chen WZ and Karmin O(1999 ) Inhibitory effect of Ginkgo biloba extract on the expression of inducible nitric oxide synthase in endothelial cells. Biochem. Pharmacol. 58:1665–1673

Crespo J, Cayón A, Fernández-Gil P, Hernández-Guerra M, Mayorga M, Domínguez- Díez A, Fernández-Escalante JC and Pons-Romero F (2001) Gene expression of tumor necrosis factor alpha and TNF-receptors, p55 and p75, in nonalcoholic steatohepatitis patients. Hepatology.34:1158-1163.

Curtis-prior P, Vere D and Fray P (1999) Therapeutic value of Ginkgo biloba in reducing symptoms of decline in mental function. J Pharm Pharmacol 51:535-541

Daniel S, Ben-Menachem T, Vasudevan G, Ma CK and Blumenkehl M (1999) Prospective evaluation of unexplained chronic liver transaminase abnormalities in asymptomatic and symptomatic patients. Am J Gastroenterol. 94:3010-3014.

Das S, Santra A, Lahiri S, Guha Mazumder DN (2005) Implications of oxidative stress and hepatic cytokine (TNF-α and IL-6) response in the pathogenesis of hepatic collagenesis in chronic arsenic toxicity. Toxicol Appl Pharmacol 204:18-26

Day CP and James OF (1998) Steatohepatitis: a tale of two "hits" ? Gastroenterology 114:842-845

Deby C, Deby-Dupont G, Dister M and Pincemail J (1993) Efficiency of Ginkgo biloba Extract(EGb761) in neutralizing ferryl ion-induced peroxidations : therapeutic implication. In: Advances in Ginkgo biloba Extract Research:Ginkgo biloba Extrxt(EGb761) as a Free-Radical Scanvenger (Ferradini C et al., ed.),Vol.2, PP.13-26. Elsevier, Paris

DeFeudis FV (1991) Ginkgo biloba extract ( Egb 761 ) Pharmacological activity and clinical applications. Paris: Editions Scientifique, Elsevier: 1-187

DeFeudis FV (1998) Ginkgo biloba extract ( Egb 761 ):from chemistry to clinic. Publr Ullestein Med and Weisbaden, Germany

Ding WX and Yin XM (2004) Dissection of the multiple mechanisms of TNF–alpha -induced apoptosis in liver injury. J Cell Mol Med. 8:445-454

Dolly M, Droy-Lefaix MT and Braquet P (1993) Retinal electrophysiology and retinotoxicity. In: Advances in Ginkgo biloba Extract Research:Ginkgo biloba Extrxt(EGb761) as a Free-Radical Scanvenger (Ferradini C et al., ed.),Vol.2, PP.66-71. Elsevier, Paris

Donnelly KL, Smith CI, Schwarzenberg SJ, Jessurun J, Boldt MD and Parks EJ (2005) Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest. 115: 1343–1351

Dugas AJ Jr, Castan˜ eda-Acosta J, Bonin GC, Price KL, Fischer NH and Winston GW (2000) Evaluation of the Total Peroxyl Radical-Scavenging Capacity of Flavonoids:
Structure-Activity Relationships. J Nat Prod. 63: 327-331

Duvnjak M, Lerotić I, Barsić N, Tomasić V, Virović Jukić L and Velagić V (2007). Pathogenesis and management issues for non-alcoholic fatty liver disease. World J Gastroenterol.13:4539-4550.
Fan JG, Xu ZJ and Wang GL (2005) Effect of lactulose on establishment of a rat non-alcoholic steatohepatitis model. World J Gastroenterol 11:5053-5056

Feldstein AE, Canbay A, Angulo P, Taniai M, Burgart LJ, Lindor KD and Gores GJ (2003) Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis. Gastroenterology 125:437-443

Folch J, Lees M and Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipids from animal tissue. J Biol Chem 226:497-509

García-Monzón C, Martín-Pérez E, Iacono OL, Fernández-Bermejo M, Majano PL, Apolinario A, Larrañaga E and Moreno-Otero R (2000) Characterization of pathogenic and prognostic factors of nonalcoholic steatohepatitis associated with obesity. J Hepatol.33:716-724.

Gardes-Albert M, Ferradini C, Sekaki A and Droy-Lefaix MT(1993) Oxygen-centered free radicals and their interactions with EGb 761 or CP 202.In: Advances in Ginkgo biloba Extract Research:Ginkgo biloba Extrxt(EGb761) as a Free-Radical Scanvenger (Ferradini C et al., ed.),Vol.2, PP.1-11. Elsevier, Paris

G´omez-Lech´on MJ, Donato MT, Mart´ınez-Romero A, Jim´enez N, Castell JV and O’Connor JE (2007) A human hepatocellular in vitro model to investigate steatosis. Chem Biol interact 165:106-116

Harputluoglu MM, Demirel U, Ciralik H, Temel I, Firat S, Ara C, Aladag M, Karincaoglu M and Hilmioglu F (2006) Protective effects of Gingko biloba on
thioacetamide-induced fulminant hepatic failure in rats. Hum Exp Toxicol 25 : 705-713

Harrison SA, Torgerson S, Hayashi P, Ward J and Schenker S (2003) Vitamin E and vitamin C treatment improves fibrosis in patients with nonalcoholic steatohepatitis. Am J Gastroenterol 98:2485–2490

Hasegawa T, Yoneda M, Nakamura K, Makino I and Terano A(2001) Plasma transforming growth factor-β1 level and efficacy of α-tocopherol in patients with non-alcoholic steatohepatitis: a pilot study. Aliment Pharmacol Ther 15: 1667-1672.

Hashizume H, Sato K, Takagi H, Hirokawa T, Kojima A, Sohara N, Kakizaki S, Mochida Y, Shimura T, Sunose Y, Ohwada S and Mori M (2007) Primary liver cancers with nonalcoholic steatohepatitis. Eur J Gastroenterol Hepatol 19:827-834.

He SX, Luo JY, Wang YP, Wang YL, Fu H, Xu JL, Zhao G and Liu EQ (2006) Effects of extract from Ginkgo biloba on carbon tetrachloride-induced liver injury in rats. World J Gastroenterol 12: 3924-3928

Huh H and Staba EJ (1992) The botany and chemistry of Ginkgo biloba. J Herbs, Spices Med Plants 1: 91-124

Jacobs BP and Browner WS (2000) Ginkgo biloba: a living fossil. Am J Med 108: 341-342

Jung HW, Chang SO, Kim CS, Rhee CS and Lim DH (1998) Effects of Ginkgo biloba extract on the cochlear damage induced by local gentamicin installation in guinea pigs. J Korean Med Sci 13:525-528

Kao JH and Chen DS (2002) Global control of hepatitis B virus infection. Lancet Infect Dis. 2:395-403

Kirsch R, Clarkson V, Verdonk RC, Marais AD, Shephard EG, Ryffel B and de la M Hall P (2006) Rodent nutritional model of steatohepatitis: Effects of endotoxin
(lipopolysaccharide) and tumor necrosis factor alpha deficiency. Journal of Gastroenterology and Hepatology 21: 174–182

Kleijnen J and Knipschild P (1992) Ginkgo biloba. Lancet 340:1136-1139

Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, Ferrell LD, Liu YC, Torbenson MS, Unalp-Arida A, Yeh M, McCullough AJ and Sanyal AJ; Nonalcoholic Steatohepatitis Clinical Research Network(2005) Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 41:1313-1321.

Kudo H, Takahara T , Yata Y, KawaiK , Zhang W and Sugiyama T (2009) Lipopolysaccharide triggered TNF-a-induced hepatocyte apoptosis in a murine non-alcoholic steatohepatitis model. Journal of Hepatology 51 : 168–175

Kudolo GB (2001) The effect of 3-month ingestion of Ginkgo bioloba extract (Egb 761) on pancreatic beta-cell function in response to glucose loading in individuals with non-insulin-dependent diabetes mellitus. Journal of Clinical Pharmacology 41: 600–611.

Lai MY (2006) Combined Interferon and RibavirinTherapy for Chronic Hepatitis C in Taiwan. Intervirology 49:91–95

Lai SW, Tan CK and Ng KC (2002) Epidemiology of fatty liver in a hospital-based study in Taiwan. South Med J 95:1288-1292

Le Bars PL and Kastelan J (2000) Efficacy and safety of a Ginkgo biloba extract. Pub Health Nutr 3: 495-499.

Le Bars PL, Katz MM, Berman N, Itil TM, Freedman AM and Schatzberg AF(1997) A placebo-controlled, double-blind, randomized trial of an extract of Ginkgo biloba for dementia. JAMA 278: 1327-1332.

Liaw YF and Chu CM (2009) Hepatitis B virus infection. Lancet. 373:582-592

Lieber CS, Leo MA, Mak KM, Xu Y, Cao Q, Ren C, Ponomarenko A and Decarli LM (2004) Model of Nonalcoholic steatohepatitis. Am J Clin Nutr 79:350-351.

Lien EJ, Ren S, Bui HH and Wang R (1999) Quantitative structure-activity relationship analysis of phenolic antioxidants. Free Radic Biol Med 26:285-294

Liu SQ, YuJP, Chen HL, Luo HS, Chen SM and Yu HG (2006) Therapeutic Effects and Molecular Mechanisms of Ginkgo Biloba Extract on Liver Fibrosis in Rats. Am J Chin Med. 34 : 99-114

Logani S, Chen MC, Tran T, Le T and Raffa RB(2000). Actions of Ginkgo biloba related to potential utility for the treatment of conditions involving cerebral hypoxia. Life Sciences 67:1389-1396

Ludwig J, Viggiano TR, McGill DB and Oh BJ (1980) Nonalcoholic steatohepatitis: Mayo clinic experiences with a hitherto unnamed disease. Mayo Clin Proc 55:434-38

Luo YJ, Yu JP, Shi ZH and Wang L(2004) Ginkgo biloba extract reverses Cl4 –induced liver fibrosis in rats. World J Gastroenterol 10:1037-1042

Mahady GB (2001) Ginkgo biloba: a review of quality, safety, and efficacy. Nutr Clin Care 4: 140-147.

Malhi H, Bronk SF, Werneburg NW and Gores GJ (2006) Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis. J Biol Chem 281:12093-12101

Marchesini G, Brizi M, Marsell-Labate AM, Bianchi G, Buqianesi E, Mccullough AJ, Forlani C and Melchionda N (1999) Association of nonalcoholic fatty liver disease with insulin resistance. Am J Med 107:450-455

Miele L, Valenza V, La Torre G, Montalto M, Cammarota G, Ricci R, Mascian`a R, Forgione A, Gabrieli ML, Perotti G, Vecchio FM, Rapaccini G, Gasbarrini G, Day CP, and Grieco1 A (2009) Increased intestinal permeability and tight junction alterations in nonalcoholic fatty liver disease. Hepatology, 49: 1877-1887

Nagai H, Matsumaru K, Feng G and Kaplowitz N (2002) Reduced glutathione depletion causes necrosis and sensitization to tumor necrosis factor-alpha-induced apoptosis in cultured mouse hepatocytes. Hepatology. 36:55-64.

Naik SR and Panda VS (2007) Antioxidant and hepatoprotective effects of Ginkgo biloba phytosomes in carbon tetrachloride-induced liver injury in rodents. Liver Int. 27 : 393-399

Neuner P, Klosner G, Schauer E, Pourmojib M, Macheiner W, Grünwald C, Knobler R, Schwarz A, Luger TA and Schwarzet T (1994). Pentoxifylline in vivo down- regulates the release of IL-1 beta, IL-6, IL-8, and tumour necrosis factor-alpha by human peripheral blood mononuclear cells. Immunology 83:262–267.

Oh MK, Winn J and Poordad F (2008) Review article: diagnosis and treatment of non- alcoholic fatty liver disease. Aliment Pharmacol Ther 28: 503–522

Oken BS, Storzbach DM and Kaye JA(1998) The efficacy of Ginkgo biloba extract on cognitive function in Alzheimer disease. Arch Neurol 55: 1409-1415.

Pener G, Kabasakal L, Yüksel M, Gedik N and Alican Y (2005) Hepatic fibrosis in biliary-obstructed rats is prevented by Ginkgo biloba treatment. World J Gastroenterol 11:5444-5449

Pessayre D, Fromenty B and Mansouri A (2004) Mitochondrial injury in steatohepatitis
Eur J Gastroenterol Hepatol 16:1095-1105

Pittler MH and Ernst E (2000) Ginkgo biloba extract for the treatment of intermittent claudication: a meta-analysis of randomized trials. Am J Med 108: 276-281.

Rapin JR, Yoa RG, Bouvier C and Drieu K(1997) Effects of repeated treatments with an extract of Ginkgo biloba (EGb 761) and bilobalide on liver and muscle glycogen contents in the non-insulin-depedent diabetic rat. Drug Development Research 40: 68–74.

Ribeiro PS, Cortez-Pinto H, Solá S, Castro RE, Ramalho RM, Baptista A, Moura MC, Camilo ME and Rodrigues CM (2004) Hepatocyte Apoptosis, Expression of Death Receptors, and Activation of NF-κB in the Liver of Nonalcoholic and Alcoholic Steatohepatitis Patients. Am J Gastroenterol 99: 1708–1717

Samuel VT, Liu ZX,Qu X, Elder BD, Bilz S, Befroy D, Romanelli AJ, and Shulman GI (2004) Mechanism of Hepatic Insulin Resistance in Non-alcoholic Fatty Liver Disease. J. Biol. Chem. 279: 32345-32353

Sanyal AJ, Campbell-Sargent C, Mirshahi F, Rizzo WB, Contos MJ, Sterling RK, Luketic VA, Shiffman ML and Clore JN (2001) Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities. Gastroenterology 120:1183-1192

Satapathy SK, Garg S, Chauhan R, Sakhuja P, Malhotra V, Sharma BC and Sarm SK (2004). Beneficial effects of tumor necrosis factor-alpha inhibition by pentoxifylline on clinical, biochemical, and metabolic parameters of patients with nonalcoholic steatohepatitis. Am J Gastroenterol 99:1946–1952.

Satapathy SK, Sakhuja P, Malhotra V, Sharma BC and Sarm SK (2007) Beneficial effects of pentoxifylline on hepatic steatosis, fibrosis, and necroinflammation in patients with nonalcoholic steatohepatitis. J Gastroenterol Hepatol 22:634–638.

Schaffner F and Thaler H (1986) Nonalcoholic fatty liver disease. Prog Liver Dis 8:283-298

Sener G, Omurtag GZ, Sehirli O¨, Tozan A, Y¨uksel M, Ercan F and Gedik N (2006) Protective effects of Ginkgo biloba against acetaminophen-induced toxicity in mice. Molecular and Cellular Biochemistry 283: 39–45

Shen J, Wang J, Zhao B, Hou J, Gao T and Xin W(1998) Effects of EGB 761 on nitric oxide and oxygen free radicals, myocardial damage and arrhythmia in ischemia– reperfusion injury in vivo. Biochem Biophys. Acta 28: 228–236, 1998

Smith JY and Luo Y(2004) Studies on molecular mechanisms of Ginkgo biloba extract. Applied Microbiology and Biotechnology 64, 465–472.

Szabo ME, Droy-Lefaix MT and Doly M(1993) Reduction of reperfusion-induced ionic
imbalance by the superoxide dismutase, vitamin E and Ginkgo biloba Extract 761 in spontaneously hypertensive rat retina. In: Advances in Ginkgo biloba Extract Research:Ginkgo biloba Extrxt(EGb761) as a Free-Radical Scanvenger (Ferradini C et al., ed.),Vol.2, PP.93-106. Elsevier, Paris

Tanaka S, Han LK, Zheng YN and Okuda H( 2004) Effects of the flavonoid fraction from Ginkgo biloba extract on the postprandial blood glucose elevation in rats. Yakugaku Zasshi 124: 605–611.

Thurman RG, Bradford BU, Iimuro Y, Knecht KT, Arteel GE, Yin M, Connor HD, Wall C, Raleigh JA, Frankenberg MV, Adachi Y, Forman DT, Brenner D, Kadiiska M and Mason RP.(1998)The role of gut-derived bacterial toxins and free radicals in alcohol-induced liver injury. J Gastroenterol Hepatol 13 (Suppl): S39-50

Varga, E, Bodi A, Ferdinandy P, Droy-Lefailax MT, Blasig LE and Tosaki A(1999) The protective effect of EGB 761 in isolated ischemic/reperfused rat hearts: a link between cardiac function and nitric oxide production. J. Cardiovasc. Pharmacol. 34: 711–717

Watanabe S, Yaginuma R, Ikejima K and Miyazaki A (2008) Liver diseases and metabolic syndrome. J Gastroenterol 43:509-518

West D.B, Boozer C.N, Moody D.L and Atkinson R.L. (1992) Dietary obesity in nine inbred mouse strains. Am. J. Physiol. 262: R1025–R1032.

Yakaryilmaz F, Guliter S, Ozenirler S, Erdem O and Akyol G (2004) Vitamin E treatment in patients with nonalcoholic steatohepatitis: A six-month, open-label study of sixteen patients. Curr Ther Res Clin Exp 65: 266–277
Yakaryilmaz F, Guliter S, Savas B, Erdem O, Ersoy R, Erden E, Akyol G, Bozkaya H and Ozenirler S (2007) Effects of vitamin E treatment on peroxisome proliferator -activated receptor-α expression and insulin resistance in patients with non-alcoholic steatohepatitis: results of a pilot study. Intern Med J 37:229-235

Yao P, Li K, Song F, Zhou S, Sun X , Zhang X, Nussler AK and Liu L (2007) Heme oxygenase-1 upregulated by Ginkgo biloba extract: Potential protection against ethanol-induced oxidative liver damage. Food Chem Toxicol 45 : 1333-1342

Younossi ZM, Gramlich T, Matteoni CA, Boparai N and McCullough AJ (2004)
Nonalcoholic fatty liver disease in patients with type 2 diabetes. Clin Gastroenterol Hepatol 2:262-265

Yuan G, Gong Z, Li J and Li X (2006) Ginkgo biloba Extract Protects against Alcohol- induced Liver Injury in Rats. Phytother. Res. 21, 234–238

Zamara E, Novo E, Marra F, Gentilini A, Romanelli RG, Caligiuri A, Robino G, Tamagno E, Aragno M, Danni O, Autelli R, Colombatto S, Dianzani MU, Pinzani M and Parola M (2004) 4-Hydroxynonenal as a selective pro-fibrogenic stimulus for activated human hepatic stellate cells. J Hepatol 40:60-80

Zhang C, Zu J, Shi H, Liu J and Qin C (2004) The effect of Ginkgo biloba extract (EGb 761) on hepatic sinusoidal endothelial cells and hepatic microcirculation in CCl4 rats. Am J Chin Med 32 : 21-31

 


若您有任何疑問,請與我們聯絡!
臺北醫學大學 圖書館 簡莉婷
E-mail:etds@tmu.edu.tw
Tel:(02) 2736-1661 ext.2519
Fax:(02) 2737-5446