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系統識別號 U0007-1704200714153946
論文名稱(中文) 牙用/骨用聚乳酸的結晶與熱裂解行為之探討
論文名稱(英文) Crystallinity and thermal degradation behavior of poly-L-lactide for dental/orthopedic use
校院名稱 臺北醫學大學
系所名稱(中) 牙醫學系碩博士班
系所名稱(英) Graduate School of Dentistry
學年度 89
學期 2
出版年 90
研究生(中文) 黃玉錤
學號 m8704002
學位類別 碩士
語文別 中文
口試日期
論文頁數 64頁
口試委員 指導教授-李勝揚
指導教授-曾 厚
指導教授-林哲堂
關鍵字(中) 聚乳酸性
生物可吸收
熱熔熱壓成型
熱性質
結晶性
關鍵字(英) Poly-L-lactide
biomaterial
hot compression molding
thermal properties
crystallinity
學科別分類
中文摘要 本研究之目的在於探討具生物分解性的聚左乳酸(poly-L-lactide,簡稱PLLA)加工過程中,溫度及環境對分子量改變和結晶行為的影響,以期在製備新型生物可吸收性材料時,能控制其機械強度與降解速率,以應用於牙科或骨科硬組織之修復。實驗採熱熔熱壓成型法,製備聚乳酸試塊,探討:(1)PLLA複合材之基本性質,以MTS測試其彎曲強度,用DSC來檢測熔點,再以測定毛細管黏度分析分子量的變化;(2)量測PLLA在溶解、再沈、乾燥、熔融和熱壓等加工過程中,其分子量的降解程度。另外,利用濕式成膜的方式,製備聚乳酸薄膜對聚乳酸的結晶性進行探討。實驗發現220 ?C為所測試聚乳酸熱熔加工的適當溫度;在不同的加溫氣氛中,聚乳酸到達融熔所需的時間有所不同,空氣下熱熔較有效率,但過程之熱性質變化較為激烈,而真空下加溫所得聚乳酸彎曲強度明顯大於在空氣中加溫。不同降溫系統亦造成不同聚乳酸彎曲強度,在氮氣下降溫的彎曲強度較空氣中的值高。亦即,於真空中熱熔並在氮氣下冷卻的聚乳酸所得之彎曲強度(115.9 + 1.3 MPa)最高,而於空氣中加溫並冷卻的聚乳酸之彎曲強度(76.9 + 1.8 MPa)最低。結晶熱焓、熔點波峰和繞射角隨著時間而變化,其影響因素包含了再結晶效應與熱裂解效應。所得聚乳酸的成型物經由適當的熱處理,可以改變聚乳酸的結晶度和晶型,藉此進一步的掌握降解速率。但對於PLLA,以射出成型之試樣經過熱處理後,彎曲強度會下降(由113.5下降至87.6 MPa),物性較脆;而藉由PDLLA的改質,經過熱處理後,彎曲強度增加(由116.8增加至146.5 MPa)和伸長率都會增加,且有適當的結晶度。本實驗對於寡乳酸聚合物的基本性質和加工條件已有初步瞭解,以射出成型所製備之聚乳酸試樣經由熱處理後的彎曲強度亦高達146 MPa,聚乳酸的機械強度和降解速率與其分子量、結晶度息息相關,因此對於升溫環境的保護,以及適當時間的再結晶處理,都是聚乳酸加工條件最佳化的重要因素。
英文摘要 Poly-L-Lactide (PLLA) was used to prepare objects as useful dental/orthopedic biomaterial in this investigation. Samples were prepared by compression molding in the study. PLLA was heated at 220 ?C in air atmosphere and nitrogen atmosphere, respectively, to achieve a molten phase for molding. The PLLA was then molded immediately by with hot press, followed by cooling in nitrogen atmosphere or in air atmosphere. PLLA film used for the annealing experiment was obtained through the solvent-casting method. Annealing of PLLA films was performed under different annealing time. The basic characterization of the prepared PLLA samples were performed by differential scanning calorimeter and X-ray diffraction pattern for thermal properties and crystallinity, and by material testing system for mechanical strength. The bending strength of samples prepared under nitrogen atmosphere (115.9 + 1.3 MPa) (or under vacuum) was better than that of samples prepared under air atmosphere (76.9 + 1.8 MPa). The thermal property of the sample prepared under nitrogen atmosphere (or under vacuum) was more stable than that of samples prepared under air atmosphere. The crystallinity and melting temperatures of PLLA film increased with annealing time. The enthalpy of crystallization, melting peak and X-ray diffraction angle were change depended on the annealing time, the effect factors included recrystallization and thermal degradation effect. The bending strength of PLLA/PDLLA specimen that prepared by injection molding and via thermal treatment was reached 146 MPa . These results suggest that better PLLA objects can be obtained under optimal conditions, as described above.
論文目次 中文摘要………………………………………………………..I 英文摘要…………………………………………………………III 目 錄 …………………………………………………………V 圖表目錄………………………………………………………..VII 第一章 緒 論……………………………………………………..1 1-1 前 言……………………………………………….1 1-2 研究目的…………………………………………..3 第二章 文獻回顧………………………………………………….4 2-1 聚乳酸………………………………………………4 2-2 PLA材料之加工方式……………………………….7 2-3 PLA材料之生物分解與機械強度控制…………….7 第三章 研究材料和方法…………………………………………10 3-1 材料與試劑……………………………………….10 3-2 儀器設備………………………………………….10 3-3 研究方法與進行步驟…………………………….12 3-3.1 聚乳酸試樣之加工製備………………….12 3-3.2 PLLA成型物之基本性質測定…………….12 3-3.3 聚乳酸結晶行為之觀察………………..15 3-3.4 乳酸單體濃度對聚乳酸薄膜熱性質的影響 …………………………………………..16 3-3.5 PLLA機械性質強化的研究…………….17 第四章 實驗結果………………………………………………..18 第五章 實驗討論………………………………………………..22 第六章 結 論…………………………………………………….29 參考文獻…………………………………….……………………30 附錄……………………………………….………………………57 圖表目錄 表一、PLLA ( mw:12KDa,ηsp=3.19 )在不同環境下加工後的物理性質之比較………………………………………………………..34 表二、PLLA ( mw:21KDa,ηsp=4.16)在不同環境下加工後的物理性質之比較……………………………………………………….34 表三、不同濃度乳酸單體的聚乳酸薄膜在空氣下以180 ℃熱處理60分鐘前後之熔點(Tm)和冷結晶溫度(Tc)變化………………35 表四、不同濃度乳酸單體的聚乳酸薄膜在空氣下以180 ℃熱處理60分鐘後之熔點熱焓(ΔHm)、結晶熱焓(ΔHc)……………..36 表五、乳酸試樣在不同溫度熱處理4小時後之彎曲強度和彈性模數 ……………………………………………………………….37 圖一、DSC thermograms of PLLA (mw:210 kDa) with different holding time at 220 ℃ under air gas flow……………………………38 圖二、DSC thermograms of PLLA (mw:123 kDa) with different holding time at 220 ℃ under oxygen gas flow……………………….39 圖三、The environment effect of bending strength and viscosity on the PLLA ( mw:120 kDa )…………………………………………40 圖四、The environment effect of bending strength and vicosity on the PLLA ( mw:210 kDa )…………………………………………41 圖五、SEM micrography of the fracture surface of PLLA (120 kDa) by prepared under four conditions………………………………..42 圖六、SEM micrography of the fracture surface of PLLA (210 kDa) by prepared under four conditions………………………………..43 圖七、DSC thermograms of PLLA (mw:120 kDa) after different annealing treatment at 105 ℃………………………………...44 圖八、X-ray diffraction pattern of PLLA (mw:120 kDa) with different annealing process………………………………………………45 圖九、DSC thermograms of PLLA with different concentration of lactide ( before heating tx.)……………………………………………46 圖十、DSC thermograms of PLLA with different concentration of lactide ( after heating tx.)………………………………………………47 圖十一、Tc & Tm change of PLLA with different concentration of lactide ……………………………………………………………….48 圖十二、Crystallinity of PLLA with different concentration of lactide ……………………………………………………………….49 圖十三、X-ray diffraction pattern of PLLA with different annealing temperature for 4 hours at N2 atmosphere………………….50 圖十四、X-ray diffraction pattern of PDLLA/PLLA with different annealing temperature for 4 hours at N2 atmosphere……….51 圖十五、DSC thermograms of PLLA (mw:123 kDa) under different gas flow at 220 ℃ for holding 60 mins……………………...52 圖十六、Chang in molecular weight (Mw) with annealing time at 105 oC…………………………………………………………..53 圖十七、force-displacement curve of PLLA with different temperature for 4 hours at N2 atmosphere…………………………………….54 圖十八、force-displacement curve of PDLLA/PLLA with different temperature for 4 hours at N2 atmosphere…………………55 圖十九、Bending strength of PLLA & PDLLA/PLLA with different annealing temperature for 4 hours at N2 atmosphere………56 圖二十、Elastic modulus of PLLA & PDLLA/PLLA with different annealing temperature for 4 hours at N2 atmosphere………57
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系統識別號 U0007-1704200714153974
論文名稱(中文) 聚左乳酸摻合及其電漿表面處理之物化性質探討
論文名稱(英文) Effects of Blending and Plasma Surface Treatment on Physical-chemical property of “Poly-L-lactic acid”
校院名稱 臺北醫學大學
系所名稱(中) 牙醫學系碩博士班
系所名稱(英) Graduate School of Dentistry
學年度 89
學期 2
出版年 90
研究生(中文) 陳欽德
學號 m88040054
學位類別 碩士
語文別 中文
口試日期
論文頁數 111頁
口試委員 指導教授-李勝揚 教授
指導教授-陳建中 助理教授
關鍵字(中) 聚左乳酸
關鍵字(英) Poly-L-lactic acid
學科別分類
中文摘要 聚左乳酸是近二十年來,高分子生物吸收性骨接合固定材料中最具代表性之一,不過,其使用最大限制在於初期機械強度快速降低,提高分子量或結晶度,雖能提昇聚左乳酸的機械強度,卻又會在水解過程中,產生大塊裂解產物及表面崩裂,且高結晶度的聚左乳酸,因具有親水性官能基極易與水分子產生鍵結,導致水解速率上昇,使得材料機械強度不易維持,所以一般在製備聚左乳酸骨釘及骨板時,必須經過加熱熔解、熱退火處理,才能獲致高機械強度的製成品。 聚左乳酸可分為左旋性的PLLA,右旋性的PDLA,右左旋性的PDLLA等三種聚左乳酸。因生物體中自然存在的乳酸皆為L form。其代謝產物可被人體吸收,故本研究選擇PLLA做為主要的實驗題材。高分子聚左乳酸 (HO(CH4COO)nH),分子量愈大,n值愈高。其結晶行為與分子鏈排列規則有關。分子鏈排列愈規則,結晶度愈高。本研究實驗重點分為兩部份,第一部份主要是探討不同分子量及不同比例的摻合對結晶行為和熔解熱焓之影響,並用實驗數據來證明其合理性。 本研究第二重點實驗是以電漿作表面改質,探討不同電漿能量條件及不同厚度疏水官能基薄膜對 PLLA 表面改質後的潑水度的高低變化,進而調節水解速率的快慢。 相同分子量的聚左乳酸,升高溫度至200℃、維持1.5hr熔解後,雖產生結構重新排列與新結晶度,但熔解前後的吸收峰皆相似,表示化學性質仍相同,聚左乳酸A(Mw17萬)的紅外線光譜.除了在主要的官能基吸收峰相似外。最大的差別在3500CM-1上有一巨大吸收峰,此表示為水分子存在,原因在於Mw17萬組購買較早,經過一段時間儲存,吸附較多水分子,經過200℃ 1.5hr熔融後吸收峰變小。因此其後所做熱微差掃瞄分析儀量測或其他的實驗,皆需昇溫到100℃以上以便防止反應過程因為水分子的存在產生水解作用。 TGA圖顯示分子量20萬的PLLA、10萬的PLLA、17萬的PLLA,其分解過程是一次崩裂,且崩裂溫度並無明顯差異。 本實驗研究結晶行為變化,最常用的大分子量為17萬和21萬,這些聚左乳酸是日本東京島津公司提供,從該公司產品目錄得知為重量平均分子量。21萬組的Mw=204157。17萬組的Mw=134312。因為17萬組購買較早,經過一段時間儲存,已經產生水解作用。因此以後購買材料需要確定儲存期。 小分子量摻合與大分子量結晶度之改變,及其結晶行為之探討。在以往文獻中聚左乳酸摻合其它高分子如 PGA 等混合來增加其結晶度是已有的,但本論文用同樣的聚左乳酸來當小分子量摻合是以往文獻所沒有的,所以本研究是一個新發現。 由實驗結果顯示,10%小分子量的摻和亦能使大分子量的熔解熱焓再回復一部份,雖然無法回復到沒有加工前之結晶度,但比破壞後再結晶度行為已達原有之 3-4 倍強。另外摻和30%、20%、10%小分子量聚左乳酸即使降溫速率較快,亦沒有多大的差別。從本實驗數據可以合理推測加工時,降溫速率可以不必做較大的考量。 另外用原始的熔解熱焓所佔比例計算,得知實驗結果比知道實驗值比估計值好,估計值會隨不同的降溫速率而改變,但是實驗值隨不同的降溫速率沒有多大的改變。所以實驗值比估計值好很多。 電漿對聚左乳酸及共聚化合物做表面處理,探討水解難易度的變化。為了降低植入初期水解速率,來維持植入初期機械強度,應用電漿技術功能中電漿表面蝕刻活化PLLA高分子基材表面,再鍍上TMDS疏水官能基薄膜,使其成為具有疏水官能基的,相信能進一步降低聚左乳酸的水解速率。本實驗除了量測撥水度的變化,進一步了解PLLA表面疏水性改質。也使用SEM及EDS利用矽吸收峰的出現確認疏水官能基的存在與否。以確認TMDS疏水官能基是否有鍍膜在PLLA表面上。 由實驗數據推測聚左乳酸應用CF4及TMDS電漿的改質,應是 PLLA經由CF4蝕刻,再產生TMDS的coating效果為最佳。 另外由實驗數據顯示是電源50W功率時,不同分子量、不同處理時間之接觸角值,電漿處理時間愈長,薄膜的接觸角度愈低,性質愈傾向於親水性。相同條件電漿鍍膜TMDS 5分鐘時以不同分子量及CF4電漿處理不同時間於PLLA表面,PLLA 表面活化及接觸角度愈低時,電漿鍍膜效應後,PLLA表面性質愈傾向於疏水性。電漿處理10分鐘時,接觸角度最低,然後再電漿鍍膜TMDS膜,接觸角度反而最高,顯示高分子基材在50W功率電漿處理時,時間愈長,PLLA接觸角度最低,因此電漿鍍膜TMDS效果愈好,並不一定需要較高電源功率。
英文摘要 Polylactic acid polymers and their copolymers have attracted attentions in maxilla and mandible bone fracture areas, due to their biocompatibility, degradability in vitro and vivo and good mechanical properties. In the past two decades, lactic acid homo- and copolymers, and their polymer blends have been intensively studied because of their significantly high hydrolyzability in the human body as well as in natural circumstances. In this study, plasma resources such as O2, CF4, TMDSwere applied to modify the surface properties of Poly-L-lactic acid with various molecular weights and PLLA/PGA (polyglycolide) for the purposes of reducing their hydrolysis rates as well as increasing adhesions with their bonding substrates. Additionally, the thermal behavior was studied to understand the interconnections of uniforming with thermally stressed materials. The studies of DSC and TGA are being focused to further understand their mechanism of crystals kinetic behavior applied in molding processing. The copolymer increased from the range 70∼90 degree to above 90 degree. It suggested that the hydrolysis rate would be reduced. The DSC results also showed that PLLA blend of 50,000 and 170,000(Molecular weight, Mv, even higher than 170,000) has higher crystallinity and hence better mechanical property.
論文目次 第一章 緒 論………………………………………………………….1 第二章 研究目的………………………………………………………2 第三章 文獻回顧………………………………………………………3 第四章 研究材料與實驗儀器原理……………………………………10 4-1 材料與試劑…………………………..………………………10 4-2 儀器設備………………………………………………..……10 4-3 實驗儀器原理………………………………………..………11 第五章 實驗結果………………………………………………………19 5-1 PLLA 基本性質量測結果………………………………..…19 5-1-1 紅外線光譜量測結果……………………………………19 5-1-2 PLLA 熱重量量測結果…………………………………20 5-1-3 凝膠滲透層析量測結果:以凝膠滲透層析儀(GPC)測定分子量…………………………………….………………21 5-2 不同分子量聚左乳酸的熔解熱焓和聚左乳酸內小分子量的摻合,對結晶行為和熔解熱焓的影響之研究…………….23 5-3 電漿表面處理對聚左乳酸及其共聚合物水解難易度變化之研究…………………………………………………………..26 5-3-1 電漿表面處理用PLLA 薄膜試片製作及結果…………27 5-3-2 電漿 flux 再現性之研究…………………………………30 5-3-2 CF4 電漿表面活化 PLLA 及 TMDS 疏水官能基鍍膜性質探討………………………………………………….30 第六章 實驗結果討論…………………………………………………37 6-1 不同分子量聚左乳酸的熔解熱焓和聚左乳酸內小分子量的摻合,對結晶行為和熔解熱焓的影響之討論……………………………37 6-2 電漿表面處理對聚左乳酸及其共聚合物水解難易度變化之討論…………………………………………………………….42 第七章 結論……………………………………………………………47 參考文獻…………………………………………………………………49
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系統識別號 U0007-1704200714264620
論文名稱(中文) 台灣烏腳病盛行地區高血壓盛行率與一氧化氮合成酵素基因多型性之關係
論文名稱(英文) The Relationship between Nitric Oxide Synthase Gene Polymorphism and Hypertension in Blackfoot Disease Hyperendemic Area in Taiwan
校院名稱 臺北醫學大學
系所名稱(中) 公共衛生學研究所
系所名稱(英) Graduate Institute of Public Health
學年度 89
學期 2
出版年 90
研究生(中文) 蔡巧姿
學號 m8808007
學位類別 碩士
語文別 中文
口試日期
論文頁數 80頁
口試委員 指導教授-薛玉梅
關鍵字(中)
砷代謝能力
高血壓
內皮的一氧化氮合成酵素基因多型性
關鍵字(英) arsenic
arsenic metabolism capability
nitric oxide synthase gene polymorphism
學科別分類
中文摘要 本研究為探討烏腳病盛行地區居民高血壓盛行率與內皮的一氧化氮合成酵素基因多型性的關係。係以烏腳病高盛行地區的嘉義縣布袋鎮好美、復興與新民三里為研究地區。民國77至78年間所有年滿30歲且每週至少居住當地五天的現住居民為研究對象,經由受過標準化訪視訓練的訪視員利用結構式問卷進行家庭訪視。問卷內容包括人口學資料、居住史、飲水史與生活習慣等變項。此外,在民國80,82與86年各進行一次健康檢查篩檢高血壓與糖尿病患者,並以真空採血器採取受檢者血液樣本。血清與白血球分離後予以-70℃冷凍貯存,以備血清中臨床生化檢查和萃取白血球的DNA,進行內皮的一氧化氮合成酵素基因多型性分析。本研究利用82年所收集研究對象的生物檢體,隨機選取有尿液砷物種資料共327人之血液樣本進行內皮的一氧化氮合成酵素基因多型性之分析。白血球萃取的DNA,利用聚合酵素連鎖反應(PCR),再使用BanⅡ酵素做限制片段長度多型性(RFLP),分析內皮的一氧化氮合成酵素基因多型性。研究結果發現,居住在烏腳病盛行地區的居民,血壓正常者與高血壓者之基因座分布並無顯著差異。在調整內皮的一氧化氮合成酵素基因型態及砷代謝能力後,年齡、性別、身體質量指數、累積砷暴露、三酸甘油脂或低密度脂蛋白膽固醇與高血壓危險對比值呈顯著正相關。在調整年齡、性別、身體質量指數與內皮的一氧化氮合成酵素基因多型性後,累積砷暴露越高且無機砷代謝百分比越高者,高血壓危險對比值顯著增加。內皮的一氧化氮合成酵素基因座為野生型者(eNOS codon 298 (wt/wt)),高血壓危險對比值與慢性砷暴露及其他傳統危險因子有顯著相關。內皮的一氧化氮合成酵素基因座為變異型異質結合子(eNOS codon 298 (wt/vt))與變異型同質結合子(eNOS codon 298 (vt/vt))者,慢性砷暴露增加與砷代謝二級指標越差者,高血壓危險對比值也增加,雖未達統計上顯著性,但可發現內皮的一氧化氮合成酵素基因多型性有變異者高血壓的危險對比值比野生型者偏高。
英文摘要 This study was conducted to explore the relationship among nitric oxide synthase gene polymorphism, arsenic methylation capability, and the prevalence of hypertension in blackfoot disease hyperendemic area in Taiwan. Three villages, Homei, Fuhsin and Hsinming of Putai Township on the southwestern coast of Taiwan Island were selected as the study area. The residents aged 30 or more years old, who lived at least 5 days a week in the villages were recruited into this study from January to February 1989. Each study subject was personally interviewed by well-trained interviewers with standardized interview techniques and with a structured questionnaire. The history of living in the arseniasis endemic area and duration of drinking artesian well water, together with life style variables including alcohol drinking, cigarette smoking and dietary habit, as well as personal and family history of disease were obtained in the interview. In addition, the study subjects received physical examination and the fasting bloods were collected. Serum and buffy coat were separated and stored in -70℃ for the analysis of clinical biochemistry indies and nitric oxide synthase gene polymorphism. The serum samples of 327 study subjects were collected from 1993 who had urinary arsenic species data were randomly recruited as study subjects in this study. DNA was extracted from buffy coat to analyze nitric oxide synthase(eNOS) gene polymorphism utilizing polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) We found no difference in the eNOS gene polymorphism between hypertension patients and normal tension controls. On the other hand, we found that age, sex, body mass index (BMI), cumulative arsenic exposure were significantly positively related with the risk of hypertension after adjustment with eNOS gene polymorphism and arsenic metabolism capability. The higher the cumulative arsenic exposure and the lower arsenic metabolism capability increase the risk of hypertension after adjustment with age, sex, BMI, eNOS gene polymorphism. In the eNOS codon 298 (wt/wt), the odds ratio of hypertension was related with age, sex, BMI, and arsenic metabolism capability. But, the eNOS codon 298 (wt/vt) and eNOS codon 298 (vt/vt) did not related. On the other hand, in the eNOS codon 298 (wt/wt), the higher the cumulative arsenic exposure and the lower arsenic metabolism capability increase the risk of hypertension after age, sex, BMI, eNOS gene polymorphism adjustment. The same result was shown in the eNOS codon 298 (wt/vt) and eNOS codon 298 (vt/vt).
論文目次 目錄 表目錄i 圖目錄i 第一章前言1 第二章文獻探討4 第一節高血壓的定義與危險因子4 第二節砷與高血壓、糖尿病及缺血性心臟病相關研究6 第三節砷代謝能力相關性研究12 第四節內皮內皮的一氧化氮合成酵素與高血壓作用機轉相關研究15 第五節內皮的一氧化氮合成酵素的作用相關研究22 第六節砷與一氧化氮相關性研究27 第三章材料與方法28 第一節研究地區28 第二節研究對象28 第三節基本資料的收集29 第四節高血壓的診斷29 第五節慢性砷暴露與砷代謝能力指標30 第六節內皮的一氧化氮合成酵素基因多型性分析方法32 第四章結果38 第一節研究對象高血壓盛行率之比較38 第二節研究對象內皮的一氧化氮合成酵素基因座之比較45 第三節研究對象高血壓盛行率與內皮一氧化氮合成酵素基因多型性、砷代謝能力與慢性砷暴露指標之多變項分析50 第四節依研究對象不同的內皮的一氧化氮合成酵素基因多型性分層分析54 第五章討論67 第六章結論70 第七章研究限制71 參考文獻72
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系統識別號 U0007-1704200714264636
論文名稱(中文) 聚乳酸在活體內的組織反應與物理性質之變化
論文名稱(英文) Tissue response and physical change of poly-L-Lactide
校院名稱 臺北醫學大學
系所名稱(中) 牙醫學系碩博士班
系所名稱(英) Graduate School of Dentistry
學年度 88
學期 2
出版年 89
研究生(中文) 白裕仁
學號 m8704003
學位類別 碩士
語文別 中文
口試日期
論文頁數 102頁
口試委員 指導教授-李勝揚
指導教授-郭倍榮
關鍵字(中) 聚乳酸
活體
組織反應
物理性質
關鍵字(英) Poly-L-Lactide
in vivo
tissue response
physical property
學科別分類
中文摘要 到目前為止,使用聚乳酸植體最大的限制仍是其強度不足,故多用於海綿骨骨折之固定,製備時可以緩慢降溫方式來提高聚乳酸結晶度,增加聚乳酸的強度,但缺少此種聚乳酸的組織學研究,且有關聚乳酸之研究多為長期觀察,缺乏對於聚乳酸植體在海綿骨骨折癒合關鍵的前五週時期之組織學及物理性質變化的探討。所以本研究的目的在探討以緩慢降溫方式所製備之聚乳酸poly (L-lactide) 在體內35天期間的組織反應及物理性質變化。使用日本島津公司重量分子量21萬之聚乳酸,以220 ℃熱熔法,在氮氣中自220 ℃緩慢降溫產生結晶度43 %的聚乳酸,並切割成2 x 6 x 25 mm3試樣,經環氧乙烯滅菌後,植入在Sprague Dawley大鼠背部皮下組織,經1天、3天、7天、21天、35天五個試驗期間,以石臘及樹脂標本之組織學觀察,評估其組織反應並以三點彎曲強度、黏度及熱性質檢測,評估其物理性質變化。滅菌前聚乳酸植體試樣三點彎曲強度為120 MPa左右,結晶度為43 %左右,經55 ℃、2.5小時環氧乙烯滅菌後,發現滅菌前後之物理性質沒有差異。就聚乳酸植體試樣組織癒合過程而言,可分為止血期、發炎期、增生期及重塑期四個時期。術後第一至三天為止血期、發炎期,是手術外傷所造成之發炎反應,炎症細胞以中性白血球為主,術後第三天,炎症細胞以單核球細胞為主,可見纖維母細胞分布。術後第七天為增生期,炎症細胞分布範圍變小,可見單核球細胞數目減少,此時可見含有許多增生纖維母細胞及血管的纖維包膜形成,由樹脂磨片標本可見纖維包膜與聚乳酸植體緊密接觸。植入後第21天為重塑期,可見纖維包膜的厚度比第7天薄,包膜內之纖維母細胞變較細長,血管數目減少。植入後第35天亦屬重塑期,纖維包膜厚度比第21天薄,包膜內之纖維母細胞及血管數目減少。所以此聚乳酸植體試樣之組織反應是以單核球細胞為主要的炎症細胞反應,植入後第七天形成纖維包膜,且隨植入時增加,纖維包膜愈成熟,在植入35天期間未見多核吞噬細胞。就聚乳酸植體試樣物理性質變化而言,經35天試驗期間其分子量沒有明顯差異,故沒有明顯降解反應發生,能維持一穩定之三點彎曲強度、黏度及熱性質,所以植入周圍組織對此聚乳酸植體試樣反應溫和,纖維包膜隨植入時間增長而愈成熟。這可能是因為具有足夠高之初始分子量且在製備時置於氮氣中緩慢降溫,聚乳酸主鏈未受製備過程產生大幅熱降解而製備時不予施壓致使材料具有適當之結晶度,雖然進一步長期降解研究是必須的,此種製備方式所得之聚乳酸植體為一可維持五週機械強度之生物鈍性材料。
英文摘要 So far the greatest limitation of using polylactide implant is insufficient strength, so it is used in fixation of sponge bone fracture mostly. To increase the crystallinity and strength of polylactide by gradually cooling from the melting temperature to room temperature, but lacking of relating histological study. Most study of polylactide were done for long term study, there were few study about histological and physical change of polylactide during the decisively initial 5 weeks of healing period of spongy bone fracture. The purpose of this study was to investigate the tissue response and physical change of annealing poly (L-lactide) during initial 35 d in vivo test. PLLA (Mw = 210000 Da.) was obtained from Shimazu chemicals, Inc. (Japan). 2 x 6 x 25 mm3 tested plates were cut from samples prepared from heating the PLLA powder at 220 oC under vacuum without pressure and gradually cooled from 220 oC until room temperature in a pot vented with nitrogen for one night. The crystallinity value of resultant samples was 43 %. These tested plates were implanted in the dorsal subcutis of S.D. rat after sterilization with ethylene oxide and removed at 1 d、3 d、7 d、21 d and 35 d intervals. These specimens were examined histologically with paraffin embedding section and resin embedding grounding section by light microscopy to evaluate tissue response. Three points bending test, viscosity and thermal properties were also examined. The bending strength and crystallinity values of samples were 120 MPa and 43 %before sterilization. There was no significant change in physical properties after 55 oC, 2.5 h ethylene oxide sterilization. As for healing progress of PLLA implantation, there were hemostatic、inflammatory、proliferating and remodeling stages. The initial three days were defined as hemostatic and inflammatory stages, caused by surgical trauma. Inflammatory cells were neutrophils predominately. The third day after implantation was inflammatory stage with mononuclear cells predominately. Fibroblasts were also noticed. The seventh day was proliferating stage, the inflammatory infiltration area and number of mononuclear cells decreased. Fibrous capsule with proliferating fibroblasts and blood vessels was found. Close contact between PLLA implant and fibrous capsule was noticed by resin grounding section. 21 d after implantation was remodeling stage, the thickness of fibrous capsule was reduced and fibroblasts became more slender. The number of blood vessels of fibrous capsule was reduced. 35 d after implantation was remodeling stage, the thickness and numbers of blood vessels and fibroblasts of fibrous capsule were reduced further. Histologically, the inflammatory cells in tissue response of PLLA implant was mononuclear cells predominately. A fibrous tissue layer was formed around the PLLA plate from 1 wk. This became more mature over time. During the experimental period, there were no multi-nuclear phagocytes. As for physical change of PLLA implant through 35 days, there was no significant difference in molecular weight and no obvious degradation occurred. Tested samples can maintain stable three points bending strength, viscosity and thermal properties. So tissue response of tested PLLA implant was mild and fibrous capsule became more mature over time. These may result from sample with enough initial molecular weight annealed under nitrogen without large degree of thermal degradation during processing and proper crystallinity by processing without pressure. Although further long term studies for degradation are needed, the PLLA implants processed by this method have promising mechanical strength and are bioinnert materials during the five weeks periods.
論文目次 誌 謝………………………………………………………………….I 中文摘要……….………………………………………………………..II 英文摘要………………………………………………………………...V 目 錄………………………………………………………………..IX 表 目 錄……………….……………………………………………..XIV 圖 目 錄….……………….……………………………….………….XV 第一章 緒論……………………………………………………………..1 1-1 研究動機目的…………………………………………………….1 1-2 文獻回顧………………………………………………………….5 1-2.1 傳統金屬固定材的優缺點…………………………………..5 1-2.2 生物可吸收性骨接合固定材………………………………..9 第二章 實驗材料與方法………………………………………………16 2-1聚乳酸植體試樣之製備…………………………………………16 2-1.1實驗材料…………………………………………………….16 2-1.2實驗儀器…………………………………………………….16 2-1.3實驗方法…………………………………………………….16 2-2聚乳酸植體試樣之三點彎曲強度、黏度、熱性質測定……….17 2-2.1實驗材料…………………………………………………….17 2-2.2實驗儀器…………………………………………………….17 2-2.3實驗方法…………………………………………………….17 2-3環氧乙烯滅菌後聚乳酸植體試樣之三點彎曲強度、黏度、熱性 質測定…………………………………………………...……….20 2-3.1實驗材料…………………………………………………….20 2-3.2實驗儀器…………………………………………………….20 2-3.3實驗方法…………………………………………………….21 2-4聚乳酸植體試樣之組織反應觀察………………………………23 2-4.1實驗材料…………………………………………………….23 2-4.2實驗儀器…………………………………………………….24 2-4.3實驗方法…………………………………………………….24 2-4.3.1 植入聚乳酸植體試樣的處理 ………………………...24 2-4.3.2植入動物的手術 ………………………………………24 2-4.3.3植入聚乳酸試樣及組織取出時間點 …………………25 2-4.3.4植入聚乳酸植體石蠟標本的製備……………………..25 2-4.3.4.1 標本取出與固定 …………………………………25 2-4.3.4.2石蠟標本切片製作 ……………………………….25 2-4.3.5植入聚乳酸植體樹脂磨片標本的製備 ………………26 2-4.3.5.1 標本取出與固定 …………………………………26 2-4.3.5.2樹脂標本磨片製作………………………………...26 2-4.3.6對照組組織標本的製備 ………………………………27 2-4.3.6.1 標本取出與固定 …………………………………27 2-4.3.6.2對照組石蠟組織標本切片之製作 ……………….27 2-5植體試樣體內試驗後之三點彎曲強度、黏度、熱性質 測定……………………………………………………………..28 2-5.1實驗材料 …………………………………………………..28 2-5.2實驗儀器 …………………………………………………..28 2-5.3實驗方法 …………………………………………………..29 2-5.3.1 植入聚乳酸植體試樣的處理 ………………………..29 2-5.3.2植入動物的手術 ……………………………………....29 2-5.3.3植入聚乳酸植體試樣之取出時間點 …………………30 2-5.3.4植入聚乳酸植體試樣機械物理性質的測定 …………30 2-5.3.4.1 體內實驗後試樣之儲存 …………………………30 2-5.3.4.2三點彎曲強度測定 ……………………………… 30 2-5.3.4.3黏度測定 ………………………………………….31 2-5.3.4.4熱性質測定………………………………………...31 2-6 實驗流程圖…………………………………………………….32 第三章 實驗結果………………………………………………………33 3-1 聚乳酸植體試樣製備之結果…………………………………...33 3-1.1試樣之製備………………………………………………… 33 3-1.2試樣之三點彎曲強度、黏度、熱性質測定……………….33 3-1.3試樣在環氧乙烯滅菌前後物理性質變化………………….33 3-2聚乳酸植體試樣之組織反應觀察 ……………………………..34 3-2.1控制組石蠟包埋組織切片之觀察………………………….34 3-2.2實驗組石蠟包埋組織切片之觀察………………………….35 3-2.3實驗組樹脂包埋組織磨片之觀察………………………….36 3-3 體內試驗後聚乳酸植體試樣之物理性質……………………...37 3-3.1黏度測定…………………………………………………….37 3-2.2三點彎曲強度測定 ………………………………………...37 3-2.3熱性質測定 ………………………………………………...37 第四章 實驗討論 ……………………………………………………39 4-1 聚乳酸植體試樣之製備………………………………………...39 4-1.1試樣之三點彎曲強度、黏度、熱性質………………….…39 4-1.2 滅菌前後試樣之物理性質…………………………………39 4-2聚乳酸植體試樣之組織反應……………………………………41 4-3體內試驗後聚乳酸植體試樣之物理性質討論…………………46 4-3.1樣本之儲存………………………………………………….46 4-3.2黏度之測定………………………………………………….46 4-3.3三點彎曲強度……………………………………………….48 4-3.4熱性質之測定……………………………………………….49 第五章 結論 …………………………………………………………51 第六章 將來研究方向…………………………………………………52 參考文獻 ………………………………………………………………95 附錄 附錄 1 : 10 % Formaldehyde solution 固定液的製備………..….…..103 附錄2 : 2 % Paraformaldehyde +2.5 % Glutaraldehyde + 0.1 M Cacodylate 固定液的製備…..…………………………..…104 附錄 3 : Stevenel ‘s blue stain & Van Gieson’s picro-fuchsin stain 的製 備……………………………………………..………………106
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系統識別號 U0007-1704200714264641
論文名稱(中文) 中國人之粒線體控制區域 (D-loop) 鹼基序列多形性的探討
論文名稱(英文) Polymorphism of the mitochondrial control (D-loop) region in Chinese
校院名稱 臺北醫學大學
系所名稱(中) 醫學檢驗生物技術學研究所
系所名稱(英) Graduate Institute of Biomedical Technology
學年度 89
學期 2
出版年 90
研究生(中文) 陳明宏
學號 M88090118
學位類別 碩士
語文別 中文
口試日期
論文頁數 60頁
口試委員 指導教授-李宏謨博士
指導教授-曾嶔元博士
關鍵字(中) 粒線體控制區域 (D-loop)
多形性
異質化
高變異頻率I
II
III
關鍵字(英) mitochondrial control (D-loop)
polymorphism
heteroplasmy
HVR-I
II
III
學科別分類
中文摘要 摘要: 粒線體DNA是細胞內獨立於核染色體外的DNA分子。相較於核染色體DNA,它具有母系遺傳,呈環狀結構,拷貝份數多,及變異性大等特點,很適合法醫刑案上個人身分鑑別時使用。過去的研究發現粒線體DNA在控制區,即D環上有較高的變異率,其中尤其是分布位置在核甘酸序列第16086到16399之間,和43到332之間的兩段具有異於平常的高變異頻率,分別稱為HVR-I及HVR-II。然而過去的研究大多侷限在這兩個區域,而且許多較早的研究是以限制酶多形性來區分型別。對這兩個變異區以外的D環其餘位置,雖然Lutz在1999年曾報告過另一高變異區HVR-III位在479到568之間,但是在這些變異區以外D環其餘的位置,及限制酶切斷位置以外的部分之核酸序列變異性仍值得作進一步探討。 我們使用PCR放大及直接定序的方法研究63位沒有血緣關係的台灣地區漢人整個粒線體DNA控制區D環的基因鹼基序列,發現了127個位置具有DNA序列的多樣性,總計有788個變異型。在已報告過的三個高變異區中,HVR-I上有72個變異位置,HVR-II有35個位置,HVR-III有13個位置,而有9個位置是在上述三個高度變異區域之外,並有叢集在16465到16527間的現象,另外還發現33個未曾有文獻報告的位置出現DNA序列多樣性。比對結果其基因歧異度 (genetic diversity) 在HVR-I是0.999,HVR-II是0.993,HVR-III是0.850與過去文獻所得結果相仿。而16465到16527間新發現之高變異區基因歧異較低為0.614。而鑑別力則分別是0.983,0.977,0.837,及0.604。 我們也發現19例的粒線體基因異質化現象,基因異質化出現在310位置,其形式為一胸腺嘧啶插入,此位置之基因異質化過去文獻中也有報告,我們的實驗發現310位置基因異質化出現的頻率約為30﹪而過去文獻報告則為13﹪。
英文摘要 Abstract: Mitochondria DNA (mtDNA) is a subcellular sequestration. It is maternally inherited and exists in a high copy number in each cell in addition to rapidly evolving. The variable sites in the control region consists of two hypervariable segments, HVR-I and HVR-II. The sequence polymorphisms in control region between individuals are useful markers for personal identification in forensic application. Although a number of population genetics studies have already published, population genetic of the Chinese population, has not yet been established satisfactorily. We performed PCR amplification and direct sequencing to investigate the polymorphisms of the D-loop in mtDNA from sixty-three unrelated Chinese in Taiwan. Total 788 polymorphisms are found distributed in 127 variable sites. Seventy-two variable sites are in HVR-I region, thirty-five variable sites in HVR-II, and thirteen variable sites in the HVR-III, also nine sites in the regions outside the above HVRs. Thirty-three novel variable sites are not been described yet. The genetic diversity of HVR-I HVR-II and HVR-III are 0.999, 0.993, 0.850 respectively and 0.614 for and region outside the HVRs. The power of discrimination of the HVR-I HVR-II and HVR-III are 0.983, 0.977, 0.837 respectively, and 0.604 for the areas outside the HVRs. There are nineteen cases in the study found with heteroplasmy at sequence of 310, which show two populations of mitochondria; one with insertion of thymine, and the other without. High frequency of heteroplasmy at sequence 310 is quite different from the previous report.
論文目次 目錄 目錄----------------------------------------------------------------------------i 圖目錄-------------------------------------------------------------------------iv 表目錄-------------------------------------------------------------------------v 縮寫表(一)----------------------------------------------------------------vi 縮寫表(二)----------------------------------------------------------------vii 英文摘要----------------------------------------------------------------------I 中文摘要----------------------------------------------------------------------III 第一章 前言-----------------------------------------------------------------1 一、粒線體的生物性質----------------------------------------------1 二、粒線體DNA的結構--------------------------------------------4 三、粒線體DNA基因變異的類型--------------------------------8 四、造成粒線體DNA基因變異頻度增加的原因--------------9 五、造成基因異質化現象的原因----------------------------------9 六、粒線體基因體研究的應用-------------------------------------11 第二章 實驗部份-----------------------------------------------------------13 一、實驗樣本----------------------------------------------------------13 二、儀器設備-----------------------------------------------------------13 三、化學試劑-----------------------------------------------------------14 第三章 實驗方法------------------------------------------------------------16 一、全血抽取DNA---------------------------------------------------16 二、DNA聚合酶連鎖反應(Polymerase chain Reaction)----17 三、純化聚合酶連鎖反應產物--------------------------------------19 四、BIG Dye Reaction-------------------------------------------------20 五、酒精沉澱-----------------------------------------------------------21 六、Loading sample 之製備-----------------------------------------21 七、製作polyacrylamide gel-----------------------------------------22 八、DNA序列分析儀-核酸自動分析儀---------------------------23 九、DNA引子之序列(DNA Primer sequences)--------------24 十、定序問題排除-----------------------------------------------------26 第四章 實驗結果 -----------------------------------------------------------33 一、標準參考序列及命名系統--------------------------------------33 二、變異分布概況-----------------------------------------------------34 第五章 討論 -----------------------------------------------------------------47 一、族群基因變異統計計算-----------------------------------------47 二、中國人直接定序與過去PCR-RFLP方式實驗資料比較--48 三、與其他族群HVR-I,HVR-II定序結果之比較-------------49 四、其他具高變異頻率位置區比較--------------------------------50 五、高變異頻率的基因學探討--------------------------------------52 六、基因異質化現象的探討-----------------------------------------53 七、STR分型方法與粒線體DNA分型方法之比較------------54 八、關於粒線體DNA結果的判斷----------------------------------55 第六章 參考文獻-------------------------------------------------------------56 圖目錄 圖一Mitochondrial DNA map--------------------------------------------5 圖二粒線體DNA 310位置出現基因異質化,BIG Dye Reaction 受干擾-----------------------------------------------------------------29 圖三粒線體DNA 310位置未出現基因異質化,反應繼續進行 --------------------------------------------------------------------------29 圖四使用反向之引子,證實粒線體DNA 310位置有基因異質 化的現象--------------------------------------------------------------30 圖五粒線體DNA 16184位置以後有poly C出現,BIG Dye Reaction停止反應---------------------------------------------------31 圖六粒線體DNA 16184位置無連續Poly C出現,BIG Dye Reaction繼續反應---------------------------------------------------31 圖七使用反向之引子16391B解決Poly C出現的問題------------32 圖八粒線體D-loop上變異性分布有集叢性分布之現象----------40 表目錄 表一粒線體與細胞核染色體轉譯編碼方式之差異------------------3 表二不同物種粒線體基因特性------------------------------------------6 表三人類粒線體基因組和細胞核基因組的比較---------------------7 表四Big dye reaction所需之引子序列 (primer sequences)--------25 表五D-loop上共有 127個位置有不同核酸序列-------------------37 表六HVR-I、HVR-II、HVR-III、Others不同鹼基序列產生變 異的種類及個數------------------------------------------------------41 表七粒線體D-loop上具變異性DNA序列的位置分布-----------42 表八各個變異區變異種類及數目---------------------------------------46
參考文獻 第六章 參考文獻 1.Anderson S., Bankier A.T., Barrell B.G., de Bi-uijn M.H.L., Coulson A.R., Drouin J., Eperon I.C., Nierlich D.P., Roe B.A., Sanger F., Schreier P.H., Smith A.J.H., Staden R., Young I.G.: Sequence and organization of the human mitochondrial genome. Nature 1981; 290: 457-465. 2.Baasner A., Schafer C., Junge A., Madea B.: Polymorphic site in human mitochondrial DNA control region sequence: population data and maternal inheritance. Foren. Sci. Int. 1998; 98: 169-178. 3.Bendall K.E., Macaulay V.A., Baker J.R., Skeys B.C.: Heteroplasmic point mutation in the Mt DNA control region. Am. J. Hum. Genet. 1996; 59: 1276-1287. 4.Brenner C. A., Barritt J. A., Willadsen S., Cohen J.: Mitochondrial DNA heteroplasmy after human ooplasmic transplantation. Fertility and Sterility 2000; 74: 573-578. 5.Budowle B., Wilson M.R., DiZinno J.A., Stauffer C., Fasano M.A., Holland M.M., Monson K.L.: Mitochondrial DNA regions HVR-I and HVR-II population data. Foren. Sci. Int. 1999; 103: 23-25. 6.Calloway C.D., Reynolds R.L., Herrin Jr G.L., Anderson W.W.: The frequency of heteroplasmy in the HVR-II region of mtDNA differs across tissue types and increases with age. Am. J. Hum. Genet. 2000; 66: 1384-97. 7.Carracedo A., Bar W., Lincoln P., Mayr W., Morling N., Olaisen B., Schneider P., Budowle B., Brinkmann B., Gill P., Holland M., Tully G., Wilson M.: DNA commission of the international society for forensic genetic: guidelines for mitochondrial DNA typing. Foren. Sci. Int. 2000; 110: 79-85. 8.Chen W., Li Y., Chen Y., Feng H.C., Fu S.B.: A study on polymorphism of mitochondrial DNA D-loop in the Han nationality in China. Chin. J. Med. Genet. 1999; 16: 247-248. 9.Elena E. J., Cavelier L., Eriksson I., Oreland L., Gyllensten U.: Human brain contains high levels of heteroplasmy in the noncoding regions of mitochondrial DNA. Proc. Natl. Acad. Sci. USA 1996; 93: 12382-12387. 10.Greenberg B.D., Newbold I.E., Sugino A.: Intraspecific nucleotide sequence variability surrounding the origin of replication in human mitochondrial DNA. Gene. 1983; 21: 33-49. 11.Hagelberg E., Sykes B.: Ancient bone DNA amplified. Nature 1989; 342: 485-488. 12.Ivanov P.L., Wadhams M.J., Roby R.K., Holland M.M., Weedn V.W., Parsons T.J.: Mitochondria DNA sequence heteroplasmy in Grand Duke of Russia Georgij Romanov establishes the authenticity of the remains of Tsar Nicholas II. Nature Genet. 1996; 12: 417-420. 13.Jazin E.E., Cavelier L., Eriksson I., Oreland L., Gyllensten U.: Human brain contains high levels of heteroplasmy in the noncoding regions of mitochondrial DNA. Proc. Natl. Acad. Sci. USA 1996; 93: 12382-12387. 14.Lee S.D., Shin C.H., Kim K.B., Lee Y.S., Lee J.B.: Sequence variation of mitochondrial DNA control region in Koreans. Foren. Sci. Int. 1997; 87: 99-116. 15.Lutz S., Weisser H.J., Heizmann J., Pollak S.: A third hypervariable region in the human mitochondrial D-loop. Hum. Genet. 1997; 101: 384. 16.Lutz S., Weisser H.J., Heizmann J., Pollk S.: Mitochondrial heteroplasmy among maternally related individuals. Int. J. Legal Med. 2000; 113: 155-161. 17.Mahler H.R.: Mitochondrial evolution: organization and regulation of mitochondrial genes. Ann. N.Y. Acad. Sci. 1981; 361: 53-75. 18.Paabo. S.: Ancient DNA: extraction, characterization, molecular cloning, and enzymatic amplification. Proc. Natl. Acad. Sci. USA 1989; 86: 1939-1943. 19.Pai C.H., Chou S.L., Tang T.K., Wei Y.H., Yang C.H., Haplotyping of mitochondrial DNA in the D-loop region by PCR:forensic application. J. Formos. Med. Assoc. 1997; 96: 73-82. 20.Parson W., Parson T.J., Scheithauer R., Holland M.M.: Population data for 101 austrian Caucasian mitochondrial DNA D-loop sequences: application of mtDNA sequence analysis to a fprensic case. Int. J. Legal Med.1998; 111: 124-132. 21.Piercy R., K.M. Sullivan, N. Benson, P. Gill: The application of mitochondrial DNA typing to the study of white Caucasian genetic identification. Int. J. Legal Med. 1993; 106: 85-90. 22.Seo Y., Stradmann-Bellinghausen B., Rittner, K.Takahama C., Schneider P. M.: Sequence polymorphism of mitochondrial DNA control region in Japanese. Foren. Sci. Int. 1998; 97: 155-164. 23.Stoneking M., Soodyall H.: Human evolution and the mitochondrial genome. Curr. Opin. Genet. 1996; 6: 731-736. 24.Szibor R., Michael M., Spitsyn V. A., Plate I., Ginter E., Krause K.: Mitochondrial D-loop 3’ (CA) n repeat polymorphism: optimization of analysis and population data. Electrophoresis 1997; 18: 2857-2860. 25.Wilson M.R., Polanskey D., Butler J., DiZinno J.A., Replogle J., Budowie B.: Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts. Bio.Techniques 1995; 18: 662-669. 26.Yoshii T., Takeda E., Akiyama K., Ishiyama 1.: Sequence polymorphism of mitochondrial DNA and its forensic application. Jpn. J. Legal Med. 1995; 49: l242-250.

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系統識別號 U0007-1704200714342337
論文名稱(中文) 染料廠員工細胞色素酵素基因型1A2、N-乙醯基轉移酵素基因型第I型與泌尿上皮細胞週期指標之相關研究
論文名稱(英文) Association of Cytochrome P-450 1A2 and N-acetyltransferase I Polymorphism with the Urothelial Cell Cycle Index in Dye Workers
校院名稱 臺北醫學大學
系所名稱(中) 公共衛生學研究所
系所名稱(英) Graduate Institute of Public Health
學年度 88
學期 2
出版年 89
研究生(中文) 劉宇真
學號 m8708008
學位類別 碩士
語文別 中文
口試日期
論文頁數 98頁
口試委員 指導教授-葉錦瑩
關鍵字(中) 聯苯胺
細胞色素酵素基因型1A2
N-乙醯基轉移酵素基因型第I型
基因多型性
泌尿上皮細胞週期指標
關鍵字(英) Benzidine
CYP1A2
NAT1
Polymorphism
Urothelial Cell Cycle Index
學科別分類
中文摘要 在癌變的過程中,個體先天的宿因及外來環境因素可能分別或共同作用於起始、促進或進展等階段。以往學界大部分只專注於環境因素的探討,然在相同環境暴露下人類卻顯現了對疾病的不同感受性。因此本研究的目的為探討這等與生俱來易感受因子,人與人之間的分子、酵素間的差異。 人體攝入芳香環胺類物質會增加膀胱癌發生的危險性已有許多流行病學資料證實。而過去台灣染料業曾大量使用染料製造過程中的中間物質聯苯胺(Benzidine),因此,本研究選定一家位於北部地區具有二十八年歷史的染料製造廠,以本國籍員工共207人作為研究對象。藉由聚合脢連鎖反應(Polymerase Chain Reaction)和限制片段長度多型性(Restriction Fragment Length Polymorphism)結果發現細胞色素酵素1A2(-2964 GaA)基因型之分佈頻率為W(G):M(A) = 72.7%:27.3%,氮-乙醯基轉移酵素基因型第一型之分佈頻率為3:4:10 :11= 23%:49%:27%:1.4%。我們希望經由分析業者細胞色素酵素1A2基因型、氮-乙醯基轉移酵素基因型第一型與尿液檢體細胞週期分佈結果作比對,以探討不同基因型對染料製造廠員工在聯苯胺暴露後發生分子及泌尿上皮細胞傷害上的影響。 本研究結果顯示帶有CYP1A2 W/W+NAT1 10/10基因型組合者其於抽煙或聯苯胺暴露下皆對泌尿細胞週期的異常具影響性,於抽煙暴露下尤其明顯,而夜尿習慣則是呈現顯著性的保護作用。以模式分析下,調整各干擾因子後仍可發現帶有CYP1A2 W/W+NAT1 10/10基因型組合者對泌尿細胞週期異常的影響,雖未達統計上顯著意義性,但仍可提供瞭解此二基因型於人體代謝芳香胺化學物質機制中所扮演的角色。
英文摘要 In the cancerization process, congenital factors and environmental factors may jointly or separately affect one into the phases of initiation, promotion and progression. Formerly, academic world just only to probe into the environment factors. But under the same exposure, human shows differential susceptibility to diseases. Therefore, our study aims to confer the variation of molecular and enzyme between man to man which inherited. There is a lot of epidemiology data supported that human being increases the probability getting bladder cancer when they were exposed to aromatic amines. In the past, Taiwan dyestuff industries used amounts of intermediate (Benzidine) in the manufacturing process. Consequently, a total of 207 workers in our study were sampled from a dyestuff manufacturing factory which established for 28 years and located at northern part of Taiwan. With the techniques of Polymerase Chain Reaction and Restriction Fragment Length Polymorphism, we discovered the genotype distribution of CYP1A2(-2964 G→A mutant) is W(G):M(A) = 72.7%:27.3% and NAT1 is 3:4:10:11 = 23%:49%:27%:1.4%. By the way of comparing genotype and DNA ploidy of urothelial cells, we hope to investigate the association between susceptible factors and urothelial cell damage in dyestuff manufacturing workers. As a result of our study exhibit those who carries genotype of CYP1A2 W/W+NAT1 10/10 would influence the DNA ploidy of urothelial cells to become abnormal under the exposure of smoking or benzidine, especially obvious in smoking group. In the modeling analyses, we still found the influence of CYP1A2 W/W+NAT1 10/10 genotype on abnormal DNA ploidy of urothelial cells after adjusted by several confounders, although it is not statistically significant, it still offers us to realize the roles of CYP1A2 and NAT1 in the mechanism of human metabolize aromatic amines.
論文目次 第一章、前言 第一節、研究背景--------------------------------------------- 第二節、研究目的--------------------------------------------- 第三節、研究架構--------------------------------------------- 第二章、文獻探討 第一節、染料的特性及製程--------------------------------- 2-1.1染料的特性------------------------------------------- 2-1.2染料的製程------------------------------------------- 第二節、聯苯胺的特性與健康危害------------------------ 2-2.1聯苯胺之特性---------------------------------------- 2-2.2聯苯胺之吸收、代謝、致癌途徑---------------- 2-2.3聯苯胺之健康危害---------------------------------- 2-2.4聯苯胺與相關癌症之流行病學研究------------- 2-2.5膀胱癌之相關危險因子 第三節、聯苯胺暴露的生物標記--------------------------- 2-3.1早期暴露效應標記-泌尿上皮細胞週期--------- 2-3.2易感性標記-CYP1A2、NAT1-------------------- 第三章、材料與方法 第一節、研究對象的選取------------------------------------ 第二節、資料的蒐集------------------------------------------ 3-2.1基本資料---------------------------------------------- 3-2.2暴露量評估----------------------------------------- 3-2.3泌尿細胞週期狀態之評估------------------------- 3-2.4尿液的採集與處理--------------------------------- 3-2.5血液的採集與處理---------------------------------- 3-2.6尿液的分析------------------------------------------- 3-2.7代謝基因型分析------------------------------------- 第三節、資料蒐集結果-------------------------------------- 第四節、資料的建檔與統計分析--------------------------- 第四章、結果 第一節、個案基本描述性資料------------------------------ 4-1.1研究個案之基本人口學資料---------------------- 4-1.2研究個案日常生活習慣、泌尿系統自覺症狀資料--------------------------------------------------- 第二節、個案基本資料之單變項分析--------------------- 4-2.1個案泌尿細胞週期值與人口學因子之單變項分析--------------------------------------------------- 4-2.2個案泌尿細胞週期值與日常生活習慣、泌尿系統自覺症狀因子之單變項分析--------------- 第三節、細胞酵素1A2基因多型性分析----------------- 4-3.1 CYP1A2基因型分析(DNA濃度適量者)------- 4-3.2 CYP1A2基因型分析(DNA濃度不足者)------- 第四節、氮-乙醯基轉移酵素第一型基因多型性分析-- 4-4.1 NAT1基因型分析(DNA濃度適量者)---------- 4-4.2 NAT1基因型分析(DNA濃度不足者)---------- 第五節、研究個案對偶基因之頻率分佈情形------------ 4-5.1 CYP1A2基因型分佈頻率分析方面------------ 4-5.2 NAT1基因型分佈頻率分析方面----------------- 第六節、研究個案對偶基因與細胞週期異常頻率之分析------------------------------------------------------ 4-6.1 CYP1A2基因型與泌尿細胞週期之分析------- 4-6.2 NAT1基因型與泌尿細胞週期之分析----------- 4-6.3 CYP1A2、NAT1基因型組合與泌尿細胞週期之分析------------------------------------------------ 第七節、研究個案對偶基因組合之分層分析------------ 4-7.1不同抽煙習慣間基因型組合之分層分析------- 4-7.2現場+非現場部門之不同抽煙習慣間基因型組合之分層分析------------------------------------ 4-7.3不同年資間基因型組合之分層分析------------- 第八節、研究個案之多變項複回歸分析------------------ 第五章、討論--------------------------------------------------------- 第六章、研究限制--------------------------------------------------- 第七章、結論與建議------------------------------------------------ 參考文獻--------------------------------------------------------------- 圖表目次 頁次 表2-2.a、聯苯胺之癌症相關研究-------------------------------- 19 表2-3.a、流式細胞儀之癌症相關研究-------------------------- 22 表2-3.b、CYP1A2酵素之癌症相關研究----------------------- 25 表2-3.c、NAT1酵素之癌症相關研究--------------------------- 27 Figure 1、芳香環胺類化學物質於人體內代謝途徑----------- 56 Figure 2、聯苯胺於人體內經由NAT1、CYP1A2酵素代謝 之途徑----------------------------------------------------- 57 表4-1.a、選取之研究個案人口學結構-------------------------- 58 表4-1.b、選取之研究個案日常生活習慣之分佈頻率-------- 59 表4-1.c、個案泌尿系統自覺症狀之分析----------------------- 60 表4-1.d、個案男女員工之連續變項分佈----------------------- 60 表4-2.a、個案泌尿細胞週期值異常與各危險因子之單變 項分析----------------------------------------------------- 61 圖4-3.a、CYP1A2-PCR--------------------------------------------- 63 圖4-3.b、CYP1A2-RFLP------------------------------------------- 63 圖4-3.c、CYP1A2-nested PCR------------------------------------ 63 圖4-3.d、CYP1A2-nested RFLP----------------------------------- 63 圖4-4.a、NAT1-PCR------------------------------------------------ 64 圖4-4.b、NAT1-RFLP----------------------------------------------- 64 圖4-4.c、A-S PCR--------------------------------------------------- 64 圖4-4.d、NAT1 nested-PCR---------------------------------------- 65 圖4-4.e、NAT1 nested-RFLP-------------------------------------- 65 表4-5.a、研究個案員工CYP1A2(-2964變異)對偶基因之 分佈頻率-------------------------------------------------- 66 表4-5.b、研究個案員工CYP1A2(-2964變異)基因型變異 頻率與其他研究之分析-------------------------------- 66 表4-5.c、研究個案員工NAT1對偶基因之分佈頻率--------- 67 表4-5.d、研究個案員工NAT1基因型變異頻率與其他研 究之分析-------------------------------------------------- 68 表4-6.a、個案CYP1A2基因型分型之泌尿細胞週期異常 頻率-------------------------------------------------------- 69 表4-6.b、個案泌尿細胞週期值異常與CYP1A2對偶基因 之單、多變項對數回歸分析-------------------------- 70 表4-6.c、個案NAT1基因型分型之細胞週期異常頻率----- 71 表4-6.d、個案泌尿細胞週期值異常與NAT1對偶基因之 單、多變項對數複回歸分析-------------------------- 72 表4-6.c、個案泌尿細胞週期異常與CYP1A2、NAT1基因 型組合之單、多變項對數回歸分析----------------- 73 表4-7.a、男性員工抽煙習慣之泌尿細胞週期G0G1值異常 與CYP1A2、NAT1基因型組合之單、多變項對 數回歸分析----------------------------------------------- 75 表4-7.b、男性員工抽煙習慣之泌尿細胞週期G0G1值異 常與CYP1A2、NAT1基因型組合之單、多變項 對數回歸分析-------------------------------------------- 76 表4-7.c、現場+非現場部門之男性員工泌尿細胞週期 G0G1值異常與CYP1A2、NAT1基因型組合之 單、多變項對數回歸分析----------------------------- 77 表4-7.d、現場+非現場部門之男性員工泌尿細胞週期 G0G1值異常與CYP1A2、NAT1基因型組合之 單、多變項對數回歸分析----------------------------- 78 表4-7.e、年資五年以下男性員工泌尿細胞週期G0G1值異 常與CYP1A2、NAT1基因型組合之單、多變項 對數回歸分析-------------------------------------------- 79 表4-7.f、年資五年以上男性員工泌尿細胞週期G0G1值異 常與CYP1A2、NAT1基因型組合之單、多變項 對數回歸分析-------------------------------------------- 80 表4-8.a、男性員工泌尿上皮細胞G0G1值異常之多變項對 數複回歸分析-------------------------------------------- 81 附錄目次 頁次 附錄一、染料製造工廠工人聯苯胺暴露程度之分組 91 附錄二、聯苯胺的物理化學特性 92 附錄三、流式細胞儀之基本構造 93 附錄四、細胞週期 94 附錄五、流式細胞儀分析之細胞週期柱狀圖 95 附錄六、NAT1基因完整序列 96 附錄七、NAT1之基因多型性 97 附錄八、CYP1A2基因5''端非轉譯區之部分序列 98
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系統識別號 U0007-1704200714352769
論文名稱(中文) 以氣相層析質譜儀分析氧化壓力指標-水楊酸鹽產物並評估多酚性化合物清除氫氧自由基之能力
論文名稱(英文) Using Salicyate Adducts as an Oxidative Stress Maker by GC/MS and Evaluate Hydroxyl Radicals Scavenging Effect of Polyphenols
校院名稱 臺北醫學大學
系所名稱(中) 藥學研究所
系所名稱(英) Graduate Institute of Pharmacy
學年度 89
學期 2
出版年 90
研究生(中文) 楊明玉
學號 M8801003
學位類別 碩士
語文別 中文
口試日期
論文頁數 69頁
口試委員 指導教授-許秀蘊
關鍵字(中) 氣相層析質譜儀
水楊酸鹽
氫氧自由基
自由基清除劑
多酚性化合物
類黃鹼素
關鍵字(英) GC/MS
salicylate
hydroxyl radical
free radical scavenger
polyphenol
flavonoid
學科別分類
中文摘要 在所有有氧生物體中,經由呼吸爆炸 (respiratory burst)所產生之超氧化陰離子自由基(superoxide anion radical)和過氧化氫(hydrogen peroxide),在螯合鐵存在下,進行Fenton’s反應,產生氫氧自由基(˙O H)。它是最具活性的氧自由基,以107-1010 M-1S-1的擴散速率(diffusion control rate)與大多數生物分子作用,引起細胞膜脂質過氧化反應(membrane lipid peroxidation),蛋白質凝集(protein aggregation)和DNA羥化反應(hydroxylation),因而瓦解細胞的功能和完整性。 由於氫氧自由基的半衰期短和反應性高,不易直接偵測,目前偵測體內氫氧自由基最有效且具特異性的方法之一是利用芳香環上羥化(aromatic hydroxylation)的方法。以水楊酸鹽(salicylate)與氫氧自由基經加成反應產生之羥化物,被廣泛的應用在體外或體內試驗,以偵測氫氧自由基之生成或評估具氫氧自由基清除作用的效力。 在體外試驗,以Fenton’s試劑所產生之氫氧自由基與salicylate反應,其生成物(2,3-DHBA及2,5-DHBA)以氣相層析質譜分析方法測定,並評估酚性化合物(如類黃鹼素;flavonoids)、維他命C、E、抗氧化劑(如probucol)等測試物之清除氫氧自由基的能力,其大小依序為quercetin (55.8%),rutin (54.7%),trolox (51.1%),thiourea (50.1%),myricetin (48.1%),luteolin (45.7%),(-)-epigallocatechin (41.9%), (-)-epicatechin (41.7%),morin (41.3%),(+)-catechin (40.6%), (+)-gallocatechin (38.8%),kaempferol (38.1%),wogonin (31.8%),liquiritin (30.9%),bacailein (30.2%),DMSO (30.2%),capillarisn (27.3%),genistein (26.9%),probucol (26.9%),β-carotene (26.6%), ascorbic acid (-),d-α-tocopherol (-)。 在體內試驗中,先以腹腔注射salicylate,觀測兔子體內受1,1,2,2-tetrachloroethane抑制cytochrome P450後,視其羥化產物2,3-DHBA及2,5-DHBA生成量之變化。
英文摘要 All aerobic organisms produced superoxide anion and hydrogen peroxide via respiratory burst. In the presence of chelated iron, these species are converted to hydroxyl radicals through a Fenton reaction . The hydroxyl radical is an extremely reactive oxidizing radical that will react with most biomolecules at diffusion-controlled rates (107-1010 M-1S-1), and cause membrane lipid peroxidation, protein aggregation, and DNA hydroxylation, thereby disrupting cellular functions and integrity. Because hydroxyl radical has a very short half-life and high reactivity, its measurement is very difficult. The trapping method with potential for specifically identifying hydroxyl radicals is aromatic hydroxylation. The method of salicylate produced hydroxylation adducts (2,3-DHBA and 2,5-DHBA) with hydroxyl radicals, was large used to assessment of the scavenging activity of hydroxyl radical both in vitro and in vivo. In vitro study, the polyphenols and others scavenged hydroxyl radicals generated by Fenton reaction. Free hydroxyl radicals were trapped by salicylate and adducts was detected by GC/MS. The scavenging activity of polyphenols decrease in the order: quercetin (55.8%) , rutin (54.7%), trolox (51.1%), thiourea (50.1%), myricetin (48.1%), luteolin (45.7%),(-)-epigallocatechin (41.9%),(-)-epicatechin (41.7%), morin (41.3%),(+)-catechin (40.6%),(+)-gallocatechin (38.8%),kaempferol (38.1%),wogonin (31.8%),liquiritin (30.9%),bacailein (30.2%),DMSO (30.2%),capillarisn (27.3%),genistein (26.9%),probucol (26.9%),β-carotene (26.6%),ascorbic acid (-),d-α-tocopherol (-)。 In vivo study, the change of the amount of 2,3-DHBA and 2,5-DHBA were observed when the rabbits were treated with IP of salicylate and induced by 1,1,2,2-tetrachloroethane to inhibit cytochrom P450.
論文目次 中文摘要………………………………………………………………1 英文摘要………………………………………………………………3 前言……………………………………………………………………5 材料與方法…………………………………………………………18 (一)試藥、儀器與實驗材料…………………………………18 (二)標準品溶液與其他試劑之配製…………………………21 (三)實驗方法…………………………………………………23 結果……………………………………………………………………29 (一)靈敏度測定、現性回歸、檢量線建立、 再現性試驗及回收率測定………………………………29 (二)清除氫氧自由基之體外試驗……………………………34 (三)清除氫氧自由基之體內試驗……………………………41 討論……………………………………………………………………50 (一)以GC/MS分析salicylate羥化產物………………………50 (二)衍生化試劑與GC/MS條件之選擇…………………………50 (三)類黃鹼素與清除氫氧自由基之SAR………………………55 (四)天然抗氧化物清除氫氧自由基之能力…………………56 (五)1,1,2,2-tetrachloroethane對兔子體內salicylate 羥化產物2,3-DHBA及2,5-DHBA之影響…………………57 結論……………………………………………………………………59 參考文獻………………………………………………………………61
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系統識別號 U0007-1704200714352772
論文名稱(中文) 青剛櫟之細胞培養株中多酚性成分分析方法的探討
論文名稱(英文) Studies on the analytical method for polyphenols from cell culture lines of Cyclobalanopsis glauca
校院名稱 臺北醫學大學
系所名稱(中) 藥學研究所
系所名稱(英) Graduate Institute of Pharmacy
學年度 89
學期 2
出版年 90
研究生(中文) 戴世傑
學號 M8801005
學位類別 碩士
語文別 中文
口試日期
論文頁數 60頁
口試委員 指導教授-許秀蘊
關鍵字(中) 青剛櫟
多酚性成分
細胞培養株
高效液相層析法
catechin
epicatechin
gallocatechin
epigallocatechin
關鍵字(英) Cyclobalanopsis glauca
polyphenols
high-performance liquid chromatography
catechin
epicatechin
gallocatechin
epigallocatechin
procyanidin-B-4
學科別分類
中文摘要 青剛櫟普遍生長在台灣800公尺以下之低海拔及平地地區,其新鮮葉中多酚性成分含量豐富,為了方便探討不同季節、地區間青剛櫟之遺傳變異大小和變異型式,以利選種、育林及復育之依據、醫療資源之開發,本實驗擬以青剛櫟新鮮的葉片、枝條、種子及其細胞培養株的萃取液,建立以高效液相層析法分析其多酚性成分,達到簡捷、穩定、精確的分析條件。 本實驗應用80%丙酮為萃取液,萃取青剛櫟及其細胞培養株中之多酚性成分,萃取液經濃縮後,先以二氯甲烷洗去低極性物質,再以通過C18 cartridge之方式濾除醣類物質,最後使用逆向層析管柱、酸性的梯度移動相條件及高感度photodiode-array偵測器之運用,得到有效且再現性良好的分析條件。 本次實驗結果,高效液相層析分析方法方面,r2均在0.999以上,最低可偵測濃度(LOD, limit of detection) 在2.5 ~ 10μM間,最低可定量濃度(LOQ, limit of quantity)在5 ~ 12.5μM間。從葉片中檢出(+)-catechin、(-)-epicatechin、(+)-gallocatechin、(-)-epigallocatechin、procyanidin-B-4及rutin;枝條及種子則檢出(+)-catechin、(-)-epicatechin、gallic acid及少量之(+)-gallocatechin;細胞培養株中之多酚性成分多數可測出(+)-catechin、(+)-gallocatechin及gallic acid。
英文摘要 Cyclobalanopsis glauca (Thunb. ex Murray) Oerst. are distributed very commen in forests from sea level to 800m throughout Taiwan island. They are rich in the polyphenolic compounds in their leaves. To study the polyphenolic components of Cyclobalanopsis glauca in different reason or area, we developed an analytical method using high-performance liquid chromatography to separate the polyphenolic compounds in the leaves, branches, seeds and it’s cell culture lines from Cyclobalanopsis glauca. An aqueous acetone (80%) were added to extract the polyphenols in Cyclobalanopsis glauca and it’s cell culture lines. After condensing, the extract was delipidized with dichloromethane and pass through C18 cartridge to filter out the sugars in the samples. Then it was chromatographed on reversed-phase column with acidic gradient mobile phase system and detected with photodiode-array. We found a well-defined, reproducible system for the separation of such polyphenols in the samples. The results shows the value of r2 for the analysis is above 0.999. The limit of detection (LOD) is in the range of 2.5 ~ 10 μM, the limit of quantity (LOQ) is 5 ~ 12.5μM. In the samples of leaves, there are (+)-catechin, (-)-epicatechin, (+)-gallocatechin, (-)-epigallocatechin, procyanidin-B-4 and rutin were detected. About branches and seeds, we found (+)-catechin, (-)-epicatechin and gallic acid. Most of the cell culture lines, (+)-catechin, (-)-epicatechin, (+)-gallocatechin were identified.
論文目次 中文摘要………………………………………………..….1 英文摘要………………………………………………..….3 前言……………………………………………………..….5 材料與方法……………………………………………….10 1.實驗材料…………………………….……………...10 1.1.試藥…………………………………………....10 1.2.儀器……………………………………………11 1.3.青剛櫟檢體……………………………………12 1.4.其他實驗器材…………………………………13 2.標準品溶液與試劑之配製…………………………16 2.1.內部標準品溶液……………………………….16 2.2.標準品溶液…………………………………….16 2.3.移動相之配製………………………………….17 3.實驗方法……………………………………………17 3.1.萃取…………………………………………….17 3.2.檢液之製備…………………………………….18 3.3.高效液相層析儀操作條件…………………….18 3.4.線性回歸試驗及檢量線之建立……………….19 3.5.濃度之計算…………………………………….19 3.6.回收率之測定………………………………….20 3.7.再現性試驗…………………………………….20 3.8.偵測極限之測定……………………………….20 結果………………………………………………………22 1.分析方法確效…………………………………….....22 2.青剛櫟新鮮檢體之分析結果…………………...…..23 3.青剛櫟細胞培養株之分析結果……………...……..23 討論………………………………………………………37 1.分析條件之選定………………………….………....37 2.分析波長之選定………………………….…………38 3.檢體之前處理…………………………….…………39 4.檢體分析………………………………….…………40 結論…………………………………………………...….49 參考文獻……………………………………………...….54
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系統識別號 U0007-1704200714352997
論文名稱(中文) Wogonin,Rutin,Quercetin 抑制脂多醣誘導發炎反應之研究
論文名稱(英文) INHIBITION OF WOGONIN, RUTIN AND QUERCETIN ON LIPOPOLYSACCHARIDE-INDUCED INFLAMMATORY RESPENOSES
校院名稱 臺北醫學大學
系所名稱(中) 生藥學研究所
系所名稱(英) Graduate Institute of Pharmacognosy
學年度 89
學期 2
出版年 90
研究生(中文) 黃鶴群
學號 M88030020
學位類別 碩士
語文別 中文
口試日期
論文頁數 49頁
口試委員 指導教授-楊玲玲
指導教授-陳彥洲
關鍵字(中) 脂多醣
誘導型一氧化氮合成酵素
環氧化酵素第二型
前列腺素E2
細胞激素
Wogonin
Rutin
Quercetin
關鍵字(英) Lipopolysaccharide
inducible nitric oxide synthase
iNOS
Cyclooxygenase-2
COX-2
Prostaglandin E2
Cytokine
Wogonin
Rutin
Quercetin
學科別分類
中文摘要 類黃酮成分(Flavonoids)存在於許多天然植物中,目前經研究已知有抗氧化、抗發炎與抗癌等功能。而本實驗即以RAW 264.7 巨噬細胞與Thioglycollate誘導巨噬細胞之in vitro模式與Balb/c小鼠in vivo模式探討天然物Wogonin、Rutin、Quercetin對於脂多醣(Lipopolysaccharide;LPS)誘導一氧化氮(Nitric oxide;NO)、環氧化酵素第二型 (Cyclooxygenase-2;COX-2)與前列腺素E2(Prostaglandin;PGE2)引起發炎反應之抑制作用。在in vitro模式中,結果發現Wogonin與Quercetin具有明顯抑制NO的功效。Rutin則較不明顯。在in vivo模式中,我們先以尾靜脈注射給藥方式投與Wogonin、Rutin與Quercetin後再注射LPS (10 mg / kg),並且在不同時間點施以眼窩採血,血液中NO含量將以Griess reaction檢測。由實驗結果得知,經LPS誘導後,血液中NO濃度呈現time dependent方式的增加,Wogonin、Rutin、Quercetin在本模式中具有明顯NO抑制作用。但三種天然物均無較明顯之COX-2、PGE2抑制反應,僅Wogonin在RAW 264.7 巨噬細胞中產生微弱的COX-2抑制作用。綜合以上結果,我們得知Wogonin、Rutin、Quercetin具有抑制LPS 誘導NO而產生抗發炎功能,並同時建立了一個可供天然物進行發炎反應研究之實驗模式。
英文摘要 Flavonids are major components of natural products and several biological activities have been explored extensively including antioxidant , anti-inflammation and anti-cancer . Our present study indicated that wogonin , rutin and quercetin showed the significant inhibition on Lipopolysaccharide(LPS)-induced inducible nitric oxide synthase(iNOS) and Cyclooxygenase-2 (COX-2), Prostaglandin(PGE2)production in RAW 264.7 macrophages. As the same part of experiment, wogonin and quercetin inhibited LPS-induced iNOS gene expression and NO production in primary macrophages. Upon intravenous injection of LPS 10 mg/kg in tail vein of Balb/c mice, blood were collected from retro-orbital sinus at various time-period after injection .The amount of NO in serum was measured by griess reaction. The results appeared that the amount of NO in serum was increased in a time dependent manner in LPS-treated mice, but not in control group. Pre-treatment of Balb/c mice with Nw-nitro-L-arginine methyl ester(L-NAME)followed by LPS treatment inhibited LPS induced NO induction. Pre-treatment of Balb/c mice with wogonin, rutin and quercetin, at the concentration, was able to suppress LPS induced NO production. However, no effect was detected on LPS-induced PGE2 production. Supression of LPS induced iNOS gene expression by wogonin and quercetin was demonstrated in RAW 264.7 macrophages, primary peritoneal macrophages and liver specimen of Balb/c mice. This study firstly provided in vivo evidences to identify the inhibitory activities of wogonin, rutin and quercetin on LPS-induced NO and PGE2 production
論文目次 考試委員名錄…………………………………………………………………...0 致謝………………………………………………………………….…………..0 目錄………………………………………………………………….…………..0 縮寫………………………………………………………………….…………..0 中文摘要………………………………………………………………….……..1 英文摘要………………………………………………………………….……..2 實驗部分 緒論……………………………………………………………………………...4 Ⅰ.前言………………………………………………………………………….6 Ⅱ.實驗材料…………………………………………………………………...16 1. 實驗動物…………………………………………………………………16 2. 細胞株……………………………………………………………………16 3. 實驗藥物…………………………………………………………………16 4. 實驗儀器…………………………………………………………………17 5. 動物實驗器材……………………………………………………………18 6. 套裝試劑…………………………………………………………………18 7. 免疫試劑…………………………………………………………………18 8. 一般化學試藥……………………………………………………………19 Ⅲ.實驗方法……………………………………………………………………...20 1. 藥物之製備…………………………………………………………………20 2. 細胞培養液製備…………………………………………………………….20 3. 一般實驗溶液製備………………………………………………………….21 4. 細胞數目之計算…………………………………………………………….24 5. Nitrite含量之測定…………………………………………………………...25 6. PGE2含量測定………………………………………………………………27 7. Western blot…………………………………………………………………..28 8. 統計資料…………………………………………………………………….29 Ⅳ.實驗結果………………………………………………………………………..30 1.Wogonin, Rutin, Quercetin對LPS誘導RAW 264.7巨噬細胞 之Nitrite含量影響…………………………………………………………..31 2. Wogonin, Rutin, Quercetin 對LPS誘導RAW 264.7巨噬細胞之 PGE2含量影響……………………………………………………………....32 3. Wogonin、Rutin、Quercetin對LPS誘導RAW 264.7巨噬細胞 iNOS與COX-2蛋白表現之抑制效果……………………………………….33 4. Wogonin、Rutin、Quercetin對thioglycollate 誘導Balb/c 小鼠 巨噬細胞之Nitrite含量抑制影響……………………………………………34 5. Wogonin、Rutin、Quercetin對thioglycollate 誘導Balb/c 小鼠 巨噬細胞之PGE2含量影響………………………………………………….35 6. Wogonin、Rutin、Quercetin對Thioglycollate 誘導Balb/c小鼠 巨噬細胞之iNOS與COX-2蛋白抑制表現效果……………………………36 7. LPS誘導之Balb/c小鼠在不同時間下血液中Nitrite含量之影響………37 8. L-NAME對LPS誘導Balb/c小鼠血液中Nitrite含量之影響…………...38 9. Wogonin , Rutin , Quercetin對LPS誘導Balb/c小鼠血液中 Nitrite含量產生之抑制效果………………………………………………..39 10. Wogonin、Rutin、Quercetin對LPS誘導Balb/c小鼠肝臟、肺臟 iNOS與COX-2蛋白表現抑制效果………………………………………..40 11. Wogonin、Rutin、Quercetin對LPS誘導的Balb/c小鼠血液中 PGE2含量之抑制效果………………………………………………………41 Ⅴ.討論……………………………………………………………………………..42 Ⅵ.結論…………………………………………………………………………….45 Ⅶ.參考文獻………………………………………………………………………..47
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Wight, N.J., Gottesdiener, K., Garlick, N. M., Atherton, C. T., Novak, S., Gertz, B. J., Calder , B . J., Calder, N . A., Cote, J., Wong, P., Dallob, A., Hawkey, C. J.(2001)Rofecoxib , a COX-2 inhibitor , does not inhibit human gastric mucosal prostaglandin production . Gastroenterology, 120 , 867-873 . Wong, W. Y. L.and Richards, J. S.(1991)Evidence for two antigenically distinct molecular weight variants of prostaglandin H synthase in the rat ovary. Mol. Endocrinol., 5, 1269-1279.

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系統識別號 U0007-1704200714395466
論文名稱(中文) 生物可吸收性的多孔質聚左乳酸/氫氧基磷灰石複合材的製備及其性質之探討
論文名稱(英文) Application of Porous Poly(L-lactide)/Hydroxyapatite Composites as Bioabsorbable Materials : Preparation and its Properties
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 88
學期 2
出版年 89
研究生(中文) 江怡雯
學號 m8702010
學位類別 碩士
語文別 中文
口試日期
論文頁數 120頁
口試委員 指導教授-曾厚
關鍵字(中) 氫氧基磷灰石
聚左乳酸
生物可吸收性
複合材料
關鍵字(英) Hydroxyapatite
Poly(L-lactide)
Bioabsorbable
Composites
學科別分類
中文摘要 本研究利用燒結的方法製作多孔性的氫氧基磷灰石(HA)燒結體,加入聚乳酸(PLLA)及由高分子量聚乳酸降解的乳酸寡聚物製作成PLLA/HA複合材,以作為硬組織生物可吸收性修補材之用。目前得到,使用砂糖及食鹽當作填充劑,以HA比砂糖比食鹽為1比1比1,5-45 kgf/cm2的壓力壓製成形,再以220°C下加熱使砂糖溶解,並且在1345°C燒結10小時的燒結物的強度以及孔隙度為最佳。高分子聚乳酸會隨著酸的濃度以及降解時間而分子量降低,分子數目上升,結果可由黏度值、GPC、DSC、FT-IR、X-ray、及接觸角的測定證實。從毒性測試的結果可知未洗過的HA對細胞造成的毒性最高,而使用氯仿洗過的HA毒性最低。由廣角X-ray的結果顯示,HA的燒結時間越久,或是成型壓力越大,其結晶程度越高。PLLA/HA複合材的機械性質測定結果顯示加入的聚乳酸濃度越高,其機械性質越強。
英文摘要 The porously sintered hydroxyapatite (HA) and poly(L-lactide) (PLLA) oligomer were used to prepare a series porous poly(L-lactide)/hydroxyapatide (PLLA/HA) composites as a biodegradable hard tissue repair material, and its physical-chemical properties were studied in this investigation. The used PLLA oligomer, mentioned above, was prepared via acidic hydrolytic reaction. The HA sinter with optimal strength and porosity could be obtained when HA was mixed with 1 of fold sucrose which was used as filler while binder and 1 of fold salt as filler, pressed under 5-45 kgf/cm2 force, dissolved the sucrose at 220°C, and then sintered under 1345°C for 10h. The all results of viscosity, gel permeation chromatography, thermal properties, IR spectra, X-ray diffraction pattern, and contact angle wholly reveal that the PLLA molecular weight decreasing with the acid amount and degradation time increasing. HA without any purification have a highest toxicity and HA washed with chloroform have a lowest toxicity against gingival fibroblast were found. The results of X-ray diffraction pattern present that the crystallinity increasing with sintered time and the pressure. The MTS data appear the more PLLA the higher mechanical strength.
論文目次 中文摘要……………………………………………………………………………. 1 英文摘要……………………………………………………………………………. 2 目錄…………………………………………………………………………………. I 圖表目次……………………………………………………………………………. II 第一章 緒論………………………………………………………………………. 3 1.1 論文回顧………………………………………………………………….. 3 1.2 研究目的………………………………………………………………….. 6 第二章 理論基礎…………………………………………………………………. 8 2.1 聚乳酸…………………………………………………………………….. 8 2.2 多孔質的氫氧磷灰石……………………………………………………..11 2.2.1 氫氧磷灰石………………………………………………………...12 2.2.2 氫氧磷灰石的燒結…………………………………...……………13 第三章 研究材料與方法…………………………………………...……………..14 3.1 材料與試劑………………………………………………...…………...…14 3.2 儀器設備…………………………………………………...……………...15 3.3 研究方法及進行步驟……………………………………...……………...17 3.3.1 氫氧磷灰石燒結體的製作…………………………………………17 3.3.2 乳酸寡聚物的製作…………………………………………………18 3.3.3 乳酸寡聚物的各種性質測定………………………………………19 3.3.4 氫氧磷灰石燒結體的性質測定……………………………………22 3.3.5 聚乳酸/氫氧磷灰石複合材的製作………………………………...25 3.3.6 聚乳酸/氫氧磷灰石複合材的性質測定…………………………...25 3.3.7 聚乳酸/氫氧磷灰石燒結體的活體外降解………………………...26 第四章 結果與討論………………………………………………………………..29 4.1 氫氧磷灰石燒結體的製作...………………………………………………29 4.2 乳酸寡聚物的各種性質測定………………………………………...……30 4.3 氫氧磷灰石燒結體的性質測定………………………………………...…39 4.4 聚乳酸/氫氧磷灰石的各種性質測定……………………………………..44 4.5 活體外降解實驗……………………………………………………...……48 4.5.1 乳酸的滴定…………………………………………………………48 4.5.2 活體外降解…………………………………………………………49 第五章 結論………………………………………………………………………..51 參考文獻……………………………………………………………………………..53 註…………………...……………………………………………………………….147 附錄…………………………………………………………………………………151
參考文獻 [1]Burchardt H.: Enneking W.F., Surg. Clinics North Am., 58, p.403, 1978. [2]刁名豪, “真骨陶瓷骨移植體”, 高雄醫學院牙醫學研究所碩士論文, 1997. [3]曾厚, “吸收性硬組織再生材之研發”, 行政院國家科學委員會專題研究計劃書, 1998. [4]黃慧平, “聚乳酸薄膜及複合材之性質研究”, 台北醫學院口腔復健醫學研究所碩士論文, 1998. [5]Nenad Ignjatovie, Simonida Tomic, Momcilo Dakic, Miroslav Miljkovie, Milenko Plavsic, Dragan Uskokovic, Synthesis and properties of hydroxyapatite/poly-L-lactide composite biomaterials, Biomaterials, 20, p.809, 1999. [6]Ruiyun Zhang, Peter X. Ma, Poly(a-hydroxyl acids)/ hydroxyapatite porous composites for bone-tissue engineering. I. Preparation and morphology, Journal of Biomedical Materials Research, 33, 446, 1999 [7]K. A. Hing, S.M. Best, W. Bonfield, Characterization of porous hydroxyapatite, Journal of Materials Science: Materials in Medicine, 10, p.135, 1999. [8]Suong-Hyu Hyon, Khosrow Jamshidi, Yoshito Ikada, Synthesis of polylactides with different molecular weights, Biomaterials, 18(22), p.1503, 1997. [9]A.G.A. Coombes, D.Major, J.M. Wood, D.J. Hockley, P.D. Minor, S.S. Davis,Resorbable lamellar particles of polylactide as adjuvants for influenza virus vaccines, Biomaterials, 19, p.1073, 1998 [10]P. Mainil Varlet, B. Rahn, S. Gogolewski, Long-term in vivo degradation and bone reaction to various polylactides, Biomaterials, 18(3), p.257, 1997. [11]Horst A. von Recum, Robert L. Cleek, Suzanne G. Eskin, Antonios G. Mikos, Degradation of polydispersed poly(L-lactic acid) to modulate lactic acid release, Biomaterials, 16(6), p.441, 1995. [12]Tsutomu Takizawa, M.D., D.Ms., Shaw Akizuki, M.D., D.Ms., Hirishi Horiuchi, M.D., Yukihiro Yasukawa, M.D., D.Ms., Foreigh body gonitis caused by a broken poly-L-lactic acid screw, The Journal of Arthroscopic and Related Surgery, 14(3), p.329, 1998. [13]”有機化學 ”, 東華出版社 [14]Y. Shikinami, M. Okuno, Bioresorbable devices made of forged composites of hydroxyapatite (HA) particles and poly-L-lactide (PLLA) : Part I. Basic characteristics, Binaterials, 20, p.859, 1999. [15]P. Ylinen, Filling of bone defects with porous hydroxyapatite reinforced with polylactide or polyglycolide fibres, Journal of Materials Science : Materials in Medicine, 5, p.522, 1994. [16] Ruiyun Zhang, Peter X. Ma, Porous poly(L-lactide)/apatite composites created by biomimetic process, Journal of Biomedical Materials Research, 33, p.286, 1999. [17]Dean-Mo Liu, Control of pore geometry on influencing the mechanical property of porous hydroxyapatite bioceramic, Journal of Materials Sciences letters, 15, p.419, 1996. [18] Dean-Mo Liu, Fabrication and characterization of poprous hydroxyapatite granules, Biomaterals, 17(20), p.1955, 1996. [19] Dean-Mo Liu, Fabricatipon of hydroxyapatiye ceramic with controlled porosity, Journal of Materials in Medicine, 8, p.227, 1997. [20]Darinn Cam, Suong-Hyu Hyon, Yoshito Ikada, Degradation of high molecular weight poly(L-lactide) in alkaline medium, Biomaterials, 16(11), p.833, 1995 [21]H. K. Koerten, J. van der Meulen, Degradation of calcium phosphates ceramics, Journal of Biomedical Materials Research, 33, p.78, 1999 [22]S. A. Bender, J. D. Bumgardner, M. D. Roach, K. Bessho, J. L. Ong, Effect of protein on the dissolution of HA coatings, Biomaterials, 21, p.299, 2000 [23]Masahiro Yoshimura, Hiroyuki Suda, Kengo Okamoto, Koji Ioku, Hydrothermal synthesis of biocompatible whiskers, Journal of Materials Science, 29, p.3399, 1994 [24]Buddy D. Ratner, Allen S. Hoffman, Frederick J. Schoen, Jack E. Lemons, “ Biomaterials Science “, Academic Press, 1996 [25]胡淑文, “ 生分解性聚乳酸作為修復關節軟骨材料之研究 “, 國立中興大學化學研究所碩士論文, 1998 [26]Lubert Stryer, “ Biomaterials “, W. H. Freeman and Company, 1995 [27]莊弘毅, “ 氫氧基磷灰石之射出成型製程及性質研究 “, 國立成功大學材料科學及工程研究所博士論文, 1996 [28]洪才益, “不同顆粒大小的氫氧基磷灰石對蝕骨細胞的影響 “, 私立中原大學醫學工程研究所碩士論文, 1998 [29]Atsushi Nakahira, Masato Tamai, Kiyoko Sakamoto, Shunro Yamaguchi, Sintering and Microstructure of Porous Hudroxyapatite, Journal of the Ceramic Society of Japan, 108[1], 99, 2000

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系統識別號 U0007-1704200714395473
論文名稱(中文) 探討Dextromethorphan對Pentylenetetrazol及gamma-hydroxybutyric acid誘發幼鼠癲癇行為之效應以及腦中bax及bcl-2基因之表現
論文名稱(英文) The effect of dextromethorphan on the pentylenetetrazol and gamma-hydroxybutyric acid induced-seizure behavior and bax and bcl-2 gene expression in the developing rat brain.
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 88
學期 2
出版年 89
研究生(中文) 王雅雯
學號 m8702020
學位類別 碩士
語文別 中文
口試日期
論文頁數 81頁
口試委員 指導教授-葉健全
關鍵字(中) 氫溴酸美索芬
癲癇
幼鼠
反轉錄聚合脢鏈鎖反應
基因表現
關鍵字(英) dextromethorphan
epilepsy
developing rat
Reverse transcription-polymerase chain reaction
gene expression
學科別分類
中文摘要 Dextromethorphan(DM) 為臨床上使用40年以上的非麻醉性止咳劑。在先前的研究發現 DM是一種NMDA(N-methyl-D-aspartate)接受體的離子通道阻斷劑。NMDA接受體是voltage-gated離子通道,當它被活化時,引起細胞膜去極化,引發鈣離子內流,導致細胞興奮性增加,而細胞內鈣離子濃度增加會進一步活化細胞的重要酵素,而引發一連串的生理或病理反應。因此DM具有拮抗NMDA接受體所引發的腦神經生理或病理的變化。過去的報告發現,DM在成鼠腦中具有抑制抽搐的作用,因此本實驗欲了解DM在發育時期的幼鼠腦中是否有同樣的作用,故以幼鼠為實驗動物,利用pentylenetetrazol (PTZ)及?-hydroxybutyric acid (GHB)誘發癲癇抽慉發作的產生,再投予DM來了解是否有抑制或減緩的效果。此外,為進一步探討持續或過劇烈的抽搐是否會影響腦部促細胞計劃性死亡基因表現的影響,我們利用反轉錄聚合脢鏈鎖反應(Reverse transcription-polymerase chain reaction;RT-PCR)的方法偵測PTZ對大腦皮質中c-fos、c-jun、bcl-2及bax mRNA的表現之影響,並且了解DM是否也具有減少因抽搐引起的腦神經細胞死亡的功用。而由實驗結果發現:(1) DM可抑制第14、30及60天大的老鼠,經PTZ所誘發的generalized clonic-tonic seizure,但對其餘的抽搐行為並無明顯的抑制作用。(2) DM可以藉由降低bcl-2/bax ratio 來抑制apoptosis的產生。因此在臨床治療的使用上,我們預期DM可為一安全、易取得並且副作用小的抗抽搐藥物,可用在較大歲數幼童之generalized clonic-tonic seizure。
英文摘要 Dextromethorphan (DM) is an effectively and widely used nonnarcotic antitussive agent for 40 years. Recent investigations have indicated that DM is an N-methyl-D-aspartate (NMDA) receptor antagonist. In particularly, DM could prevent or inhibit the NMDA receptor-mediated neuropathology including hypoxia-ischemia neurotoxicity and seizure activity. DM produces these effects by blocking the NMDA receptor coupled ion channel. However, most of the studies were conducted on adult animals and adult patients. The anticonvulsant effect of DM on the seizure activity occuring during the developing age is not yet well determined. We would like to investigate whether DM can effectively attenuate the chemical-induces seizure and brain damage in developing rats. The chemical materials used to induced seizure are pentylenetetrazol (PTZ) and gamma-hydroxybutyric acid (GHB), which induce absence-like seizure and generalized clonic-tonic seizure. Otherwise, the effect of DM on brain damage from PTZ and GHB-induced seizure in developing rat were investigated herein. The phenomenon were studied using the Reverse transcription-polymerase chain reaction (RT-PCR) to detect c-jun, c-fos, bcl-2 and bax mRNA expression. Our results showed that (1). In animal seizure models, DM attenuated the generalized clonic-tonic seizure of PND (post-natal day) 14, 30 and 60 rats. (2). In RT-PCR experiments, DM would inhibit apoptosis in lowing bcl-2/bax ratio. The results of our studies suggest that DM have anticonvulsant effect in treating generalized clonic-tonic seizure of PND 30 and 60 rats. And we can use DM to cure generalized clonic-tonic seizure in older children.
論文目次 目錄 頁數 致謝………………………………………..………..…………….......I 中文摘要………………………………………………….…………......II 英文摘要………………………………………………….…………......IV 圖表目次…………………………………………………………….......VI 壹、前言………………………………………………….…………......1 貳、實驗目的…………………………………….………………………..11 參、研究材料與實驗方法……………………………….………………..12 肆、實驗結果與分析…………………………………….………………..24 伍、討論…………………………………………………………………...36 陸、參考資料……………………………………………………………...42 圖表附錄…………………………………………………………………...48
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系統識別號 U0007-1704200714395478
論文名稱(中文) 脂多醣體對於Pentagastrin誘導胃酸分泌的作用:經由一氧化氮與第一型遲緩激?基因表現之探討
論文名稱(英文) Effect of Lipopolysaccharide on Pentagastrin-induced Gastric Acid Secretion:Involvement of Nitric Oxide and Bradykinin B1 mRNA
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 88
學期 2
出版年 89
研究生(中文) 郭淑娟
學號 m8702027
學位類別 碩士
語文別 中文
口試日期
論文頁數 85頁
口試委員 指導教授-蔡麗雪 博士
關鍵字(中) 脂多醣體
合成胃泌素
一氧化氮
遲緩激?
誘導性一氧化氮合成?
第一型遲緩激?受體基因表現
西方墨點法
反轉錄聚合?連鎖反應
關鍵字(英) Lipopolysaccharide
pentagastrin
nitric oxide
bradykinin
induced nitric oxide synthase
B1 bradykinin receptor
western blot
RT-PCR
學科別分類
中文摘要 中文摘要 脂多醣體(Lipopolysaccharide;LPS)又名內毒素(endotoxin),係來自革蘭氏陰性桿菌細胞壁之具有毒性磷酸化醣脂質的衍生物。本研究的目的是探討LPS在胃內所扮演的角色,及其參與胃酸分泌的作用機轉,同時並探討一氧化氮及第一型遲緩激?受體基因表現(bradykinin B1 mRNA)的情形。 結果顯示:LPS (mg/kg)可以抑制經由合成胃泌素(8 mg /kg/h)誘發胃酸分泌作用,此抑制作用可被[Des-Arg10] HOE 140, (H-158, 20 mg/kg)第一型遲緩激?受體的拮抗劑,或L- NG-nitro arginine methyl ester (L-NAME) (5 mg/kg)一氧化氮合成?抑制劑所拮抗。而靜脈注射LPS (1 mg/kg)可明顯抑制自發性胃酸分泌之作用,但L-NAME (5 mg/kg)可顯著增加自發性胃酸分泌的作用。H-158 (20 mg/kg)則對於自發性胃酸分泌的作用沒有影響,但對血液中一氧化氮含量則有顯著的減少。測量血液中一氧化氮含量顯示LPS對於合成胃泌素誘導酸分泌確可增加血漿中一氧化氮含量約兩倍左右。進一步利用西方墨點法 (Western blot)進行誘導性一氧化氮合成? (induced nitric oxide synthase;iNOS)蛋白質的表現,結果顯示LPS可隨劑量依存性及時間依存性增加誘導性一氧化氮合成?。更進一步利用反轉錄聚合?連鎖反應(Reverse Transcription Polymerase Chain Reaction;RT-PCR)的結果顯示,在胃部Bradykinin B1 mRNA之表現亦是隨著時間變化或劑量依存性而增加。 綜觀上述結果LPS在胃部的作用是抑制自發性胃酸分泌及合成胃泌素誘導胃酸分泌之作用,此一機轉可能經由一氧化氮的產生及bradykinin B1 mRNA表現來參與調控。 關鍵詞:脂多醣體、合成胃泌素、一氧化氮、遲緩激?、誘導性一氧化氮合成?、第一型遲緩激?受體基因表現、西方墨點法、反轉錄聚合?連鎖反應
英文摘要 英文摘要 Lipopolysaccharides (LPS), also termed endotoxins, are a family of toxic phosphorylated glycolipids derived from the cell envelope of gram—negative bacteria. In the present study, we attempted to evaluate the effects of LPS on gastric acid secretion:involvement of nitric oxide and bradykinin B1 mRNA. LPS (1 mg/kg) reduced pentagastrin (8 mg/kg/h)-stimulated gastric acid secretion. The inhibitory effect of LPS on pentagastrin stimulated gastric acid secretion was blocked by a potent bradykinin B1 receptor antagonist, [Des-Arg10] HOE 140 (H-158) of 20 mg/kg, or a NO synthase (NOS) inhibitor, NG-nitro arginine methyl ester, L-NAME of 5 mg/kg. LPS significantly decreased spontaneous acid secretion, but L-NAME (5 mg/kg) significantly increased the spontaneous acid secretion. H-158 did not affect the spontaneous acid secretion. H-158 was found to decrease plasma NO in spontaneous acid secretion. LPS was found to increase plasma NO production by two folds while LPS decreased pentagastrin-stimulated acid secretion. Furthermore, Western blot and RT-PCR were performed for iNOS protein and bradykinin B1 gene expression, respectively. LPS-treatment increased iNOS protein and bradykinin B1 mRNA in stomachs in a dose-dependent manner. These results suggest that LPS suppressed pentagastrin stimulated acid secretion via the production of NO. NO might play an important role in the regulation of acid secretion at least by an involvement of bradykinin B1 receptors. Key words:Lipopolysaccharide;pentagastrin;nitric oxide;bradykinin;induced nitric oxide synthase ;B1 bradykinin receptor;western blot ;RT-PCR
論文目次 目錄 認可 授權書 致謝 圖目錄 表目錄 中文摘要……………………………………………………….1 英文摘要…………………………………………………..…...3 第一章 緒論…………………………………………………...5 第二章 文獻查證…………………………………………….10 第三章 研究方法與材料…………………………………….20 第一節活體胃酸分泌實驗……………………………….21 第二節組織蛋白質製備………………………………….24 第三節蛋白質濃度分析………………………………….25 第四節SDS-PAGE 蛋白質電泳、西方墨點法…………26 第五節血液中NO含量測定…………………………….29 第六節反轉錄聚合?連鎖反應………………………….31 6-1組織RNA的製備………………………………..31 6-2訊息RNA的反轉錄作用………………………..32 6-3聚合?連鎖反應………………………………....33 第七節實驗藥品與試劑………………………………….34 7-1藥品與試劑……………………………………………34 7-2 材料(primer)…………………………………….36 7-3 溶液……………………………………………..37 第八節 影像及統計分析………………………………..40 第四章 實驗結果…………………………………………….41 第五章 討論………………………………………………….50 第六章 參考文獻…………………………………………….60 第七章 附錄………………………………………………….86 圖目錄 圖一、脂多醣體(LP)與遲緩激?受體拮抗劑(H-158)對於自發性酸分泌量的影響………………………………………….71 圖二、脂多醣體(LPS)及一氧化氮合成?抑制劑(L-NAME)對於自發性酸分泌量的影響………………………………….72 圖三、脂多醣體(LPS)與遲緩激?受體拮抗劑(H-158)對於合成胃泌素(pentagastrin)誘發胃酸分泌的影響……………...73 圖四、脂多醣體(LPS)及一氧化氮合成?抑制劑(L-NAME)對於合成胃泌素(pentagastrin)所誘發胃酸分泌的影響……...74 圖五、脂多醣體(LPS)劑量依存性與合成胃泌素(pentagastrin)誘發胃酸分泌的關係……………………………………….75 圖六、合成胃泌素(pentagastrin)刺激胃酸分泌時投與脂多醣體(LPS)對於血漿中一氧化氮(NO)含量的影響………….. 76 圖七、投與脂多醣體(LPS)之時間變化與血漿中一氧化氮(NO)含量的影響……………………………………………….77 圖八、投與脂多醣體(LPS)之時間變化與胃中誘導性一氧化氮合成?(iNOS)之相關性…………………………………….78 圖九、投與脂多醣體(LPS)之時間變化與胃中誘導性一氧化氮合成?(iNOS)密度之相關性………………………….…….79 圖十、投與脂多醣體(LPS)之劑量變化與血漿中一氧化氮(NO)含量的影響……………………………………………….80 圖十一、投與脂多醣體(LPS)之劑量變化與胃中誘導性一氧化氮合成?(iNOS)之相關性……………………………….81 圖十二、投與脂多醣體(LPS)之劑量變化與胃中誘導性一氧化氮合成?(iNOS)密度之相關性………………………….82 圖十三、投與脂多醣體(LPS)之時間變化與胃中bradykinin B1基因表現………………………………………………….83 圖十四、投與脂多醣體(LPS) 劑量變化與胃中bradykinin B1基 因表現……………………………………………….…84
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European Journal of Pharmacology 1998;348:1-10. 49.Kaplan AP, Joseph K, Shibayama Y, Nakazawa Y, Ghebrehiwet B, Reddigari S, Silverberg M: Bradykinin formation. Plasma and tissue pathways and cellular interactions. Clinical Reviews in Allergy & Immunology 1998;16:403-429. 50.Calixto JB, Medeiros YS: Bradykinin-induced biphasic response in the rat isolated stomach fundus: functional evidence for a novel bradykinin receptor. Life Sciences 1992;50:L47-L52. 51.Regoli D, Drapeau G, Rovero P, Dion S, Rhaleb NE, Barabe J, D'Orleans-Juste P, Ward P: Conversion of kinins and their antagonists into B1 receptor activators and blockers in isolated vessels. European Journal of Pharmacology 1986;127:219-224. 52.Hutcheson IR, Whittle BJ, Boughton-Smith NK: Role of nitric oxide in maintaining vascular integrity in endotoxin-induced acute intestinal damage in the rat. 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系統識別號 U0007-1704200714395487
論文名稱(中文) 治療性用藥對粘多糖體誘發大白鼠敗血性休克的抑制作用
論文名稱(英文) Inhibitory effect of therapeutic agent in LPS-induced septic shock in rats
校院名稱 臺北醫學大學
系所名稱(中) 醫學科學研究所
系所名稱(英) Graduate Institute of Medical Sciences
學年度 88
學期 2
出版年 89
研究生(中文) 李居仁
學號 m8702006
學位類別 碩士
語文別 中文
口試日期
論文頁數 33頁
口試委員 指導教授-吳志雄
指導教授-許準榕
關鍵字(中) 一氧化氮
敗血症休克
黏多醣
關鍵字(英) nitric oxide (NO)
septic shock
lipopolysaccharide (LPS)
學科別分類
中文摘要 敗血症(Septicemia),可由革蘭氏陰性菌(Gram negative bacteria)細胞壁上結構物質-黏多醣(lipopolysaccharide,LPS)所引起,藉由一氧化氮(NO)的生成,使血壓急劇下降和器官灌流不足時,引致敗血症休克(septic shock),嚴重時可致造成死亡。本實驗之目的是要探討在臨床上常用於治療敗血症的數種藥物,可能的作用機轉;藉由注射老鼠LPS模擬敗血症前,先給予Minocycline,Clindamycin,Cyclosporin,Platonin等藥物治療,評估藥物的改善狀況,同時觀察血壓、心跳、動物存活時間及偵測體內NO的生成量,來比較給葯與否之差異,且針對變化的可能原因來探討。 首先,選用四環素中的Minocycline(10mg/kg)此藥能與細菌體內核糖體30S結合,可抑制細菌蛋白質的合成。結果發現,Minocycline會改善LPS引起的低血壓現象,同時,降低體內NO的生成;此乃可能因Minocycline抑制NO的合成,以致血壓不會受LPS的作用影響。Clindamycin與Minocycline同樣會與細菌體核糖體30S結合,抑制蛋白質的合成。Clindamycin(10mg/kg),可抑制LPS所誘發的低血壓,且體內NO的合成亦同時被抑制。由此可知Minocycline與Clindamycin抗菌機轉類似,其改善LPS所誘發的低血壓現象,也具有相當的療效。Cyclosporin是一種免疫抑制劑,可抑制T--細胞所促成反應發生,並會抑制Lymphokine產生及釋放。在給LPS後30分鐘,隨即給予Cyclosporin(15mg/kg),實驗顯示,Cyclosporin並不影響LPS誘發的低血壓且NO的生成量仍有增加的趨勢,因此Cyclosporin不能改善LPS引起的低血壓作用,可能與Cyclosporin無法法抑制NO的產生量有關。Platonin為一種細胞感光色素,具有消炎及組織再生作用。在給予Platonin(10mg/kg)治療後,LPS的低血壓作用獲得改善,此時NO的合成也被減緩。由實驗證實LPS造成的低血壓反應可以給予Minocycline,Clindamycin,Platonin被改善,主要的可能原因為Minocycline等三藥會抑制NO的生合成,導致緩解敗血症休克的低血壓現象。Cyclosporin則無法改變LPS的降血壓作用,亦不會改變體內NO的合成。 在心跳方面,LPS注射至老鼠體內後,因血壓逐漸下降,導致產生代償性心搏加速。在給予Minocycline,Clindamycin,Platonin後,因體內NO合成量減少,血壓上升,使得心跳加速的現象減緩。Cyclosporin因無法改善LPS的血壓下降,以致心跳持續加快。另外,在給予Minocycline,Clidamycin,Platonin後,雖有改善LPS造成的低血壓現象,但仍然沒有增加老鼠的存活時間,因此,推測可能與LPS引起的發燒反應影響下視丘的體溫調控,因Minocycline等三藥物不能改變體溫的調節,導致體溫上升,動物死亡。再者,Minocycline等三藥可降低NO的產生,但尚無法獲知體內其他傷害性介質的變化的狀況。 綜合以上的結果,Minocycline,Clindamycin,Platonin能改善LPS誘發的低血壓作用,減緩心搏過速現象同時可降低體內NO的合成。Cyclosporin則無法治療LPS的低血壓現象,亦不會改變體內NO的合成。不管藥物是否可改變LPS的低血壓狀況,皆無法延長老鼠的存活時間。所以,由本實驗可應用在臨床上對於敗血症休克時,選用治療藥物的一項參考指標。並且,期望在敗血症發生的早期投予適當的藥物。預防併發症的產生。
英文摘要 Abstract Background Septicemia is generally caused by a cell wall component in Gram negative bacterias called lipopolysaccharide (LPS). It promotes the synthesis of nitric oxide (NO), which induces drastic blood pressure fall, tissue hypoperfusion, (a state known as “septic shock”), and even death. Objective and Methods To evaluate mechanisms of drugs commonly used in the treatment of septicemia, giving antibiotics including Minocycline (10 mg/kg), Clindamycin (10 mg/kg), Cyclosporin (15 mg/kg) and Platonin (10 mg/kg) to septicemic rat injected with LPS. Clinical improvement, blood pressure, heart rate, survival time are assessed, and the amount of endogenous NO production is determined. Difference between whether or not drug therapy was given, and possible causative factors were discussed. Results First of all, we choose minocycline (10 mg/kg), which has the ability of binding to the 30S of the ribosomes in the cytoplasm of the bacterial cell, inhibiting the synthesis of bacterial proteins. As a result, Minocycline blocks the hypotensive effect induced by LPSs, and at the same time, decreases the production of endogenous NO. Therefore, Minocycline’s blood-pressure stabilizing effect may be due to its inhibitory mechanism to the synthesis of NO. Clindamycin has the same affinity of binding to the 30S in bacterial ribosome. Giving this drug at the dose of 10 mg/kg, LPS-induced hypotension and the endogenous production of NO were effectively blocked simultaneously. We can conclude that both minocycline and clindamycin posses the same antimicrobial mechanism, effectively reversing the hypotensive phenomena induced by LPSs. Cyclosporin on the other hand, is a potent immunologic agent, acting as inhibitor of T-cell reactions, and of lymphokine production and its delivery. Our experiment showed that the administration of Cyclosporin (15 mg/kg) 30 minutes before LPS injection did not affect LPS-induced hypotension, and the production of NO even increased. Consequently, Cyclosporin’s inability of controlling LPS-induced hypotension is possibly due to its lack of inhibitory properties in NO synthesis. Platonin , a cyanine photosentizing dye, is a cellular photosensitive substance with anti-inflammatory and tissue regeneration effects. Its administration (10 mg/kg) also effectively controls the LPS-induced hypotension, and slows down the generation of NO free radicals. Other than causing hypotension, LPS induces compensatory tachycardia. By giving Minocycline, Clindamycin and Platonin, NO radicals production decreases, the blood pressure climbs up, therefore the heart rate slows down. Since Cyclosporin was unable to control LPS-induced hypotension, tachycardia persists. Although Minocycline, Clindamycin and Platonin were effective in controlling LPS-induced hypotension, survival time did not increase. This might be related to reactive fever caused by hypothalamic LPS stimulation, because these agents could not regulate body temperatures, leading to animal’s hyperthermia and death. Moreover, other biological mediators may have influential effects on decreasing the production of NO radicals. These three drugs have no effects upon other harmful substances despite decreasing the production of NO radicals. Conclusion Minocycline, Clindamycin and Platonin can effectively control LPS-induced hypotension, compensatory tachycardia, by reducing NO radicals production. Cyclosporin though, has no preventive effect against LPS-induced hypotension, due to its lack of ability in slowing down the production of NO radicals. Regardless of whether or not these drugs control LPS-induced hypotension, survival length of laboratory animals showed no improvement. The present experiment offers some parameters for choosing an effective therapeutic agent in the treatment of septicemia, especially in its early stages, in order to prevent the occurrence of complications.
論文目次 目錄 目錄…………………………………………………………..Ⅰ 表目錄………………………………………………………..Ⅱ 圖目錄………………………………………………………..Ⅲ 中文摘要……………………………………………………..Ⅳ 英文摘要……………………………………………………..Ⅵ 前言…………………………………………………………..1 材料與方法…………………………………………………..5 結果…………………………………………………………..10 討論…………………………………………………………..25 結論…………………………………………………………..29 參考文獻……………………………………………………..30
參考文獻 參考文獻 Amin, A. R., Attur, M. G., Thakker, G. D., Patel, P. D., Vyas, P. R., Patel, R. N., Patel, I. R., and S. B. Abramson. A novel mechanism of action of tetracyclines:effects on nitric oxide synthase. Proc. Natl. Acad. Sci. 1996; 93 (24):14014-9. Attur, M. G. Patel, R. N., Patel, P. D. Abramson, S. B. and A. R. Amin. Tetracycline up-regulates COX-2 expression and prostaglandin E2 production independent of its effect on nitric oxide. J. Immunol .1999;162(6):3160-7. Busse R, and A. Mulsch. Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin. FEBS Lett. 1990;2665 :133-136. Cocks, T. M., J. A. Angus, J. H. Campbell and G. R. Campbell. Release and properties of endothelium-derived relaxing factor (EDRF) from endothelial cells in culture. J Cell Physiol 1985;123:310-320. Dalnogare AR:Septic shock. Am. J. Med . Sci.1991;302:50-65. Dinarello, C. A., Gatti, S., & Bartfai, T.. Fever:links with an ancient receptor. Cur. Biol. 1999;9, R147-R150. Griffiths, M. J. D., M. Messent, R. J. Macallister, and T. W. Evans. Aminoguanidine selectively inhibits inducible nitric oxide synthase. Brit. J. Pharmacol. 1993;110:963-968. Halushks, P. V., Reines, H. D., Barrow, S. E., Blair, I. A., Dollery, C.T., Rambo, W., Cook, J. A. and Wise, W. C. Elevated plasma 6-keto-prostaglandin F1 alpha in patients in septic shock. Crit. Care Med. 1985; 13:451-453. Hort, E. C. and Penfold, W. J. The relation of salvation fever to other forms of injection fever. Proc. Of Royal Soc. Medi. 1992; 5:131-139. Kilbourn, R. G. and Griffith, O. W.:Overproduction of nitric oxide in cytokine-mediated and septic shock. J. Natl. Cancer Inst. 1992; 84:827-831. Kilbourn, R. G., S. S. Gross., A. Jubran, J. Adams, O. W. Griffith, R. Levi, and R. F. Lodato. NG-methyl-Largine inhibits tumor necrosis factor-induced hypotension:implications for the involvement of nitric oxide. Proc. Natl. Acad. Sci. 1990;87:3629-3632. Moncada, S., R. M. J. Palmer, and E. A. Higgs. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol. Rev . 1991;43: 109-142. Nakagawa, Y., Homma, S., Yamamoto, I., Banno, M., Nakazato, H. and Yamamoto, N. In vivo and in vitro activation of macrophages with a cyanine photosensitizing dye, platonin. Cancer Immunol. Imm. 1993;37(3):157-62. Oyanagui, Y. Platonin, a photosensitizing cyanine dye, suppresses acute inflammation. Archives Internationales de Pharmacodynamie et de Therapie. 1983; 266(1):162-72. Rietschel, E. T. and Brade, H. Bacterial endotoxins. Sci. Am. 1992;267,54-61. Seibert, F. Fever producing substance found in some distilled waters. Am. J. Physiol. 1923;67, 90-104. Smith, L. H., and Their, S. O.Pathophysiology:The Biological Principles of Disease:W. B. Saunders Company. 1985;p. 64-165 . Szabo, C., Mitchell, J. A., Thiemermann, C., and Vane, J. R. Nitric oxide-mediated hyporeactivity to noradrenaline precedes the induction of nitric oxide synthase in endotoxin shock. Br. J. Pharmacol. 1993;108:786-92. Whiteman, M. and Halliwell, B. Prevention of peroxynitrite-dependent tyrosine nitation and inactivation of alphal-antiproteninase by antibiotics. Free Radical Research. 1997; 26(1):49-56. Wright, C. E., Rees, D. D., and Moncada, S.:Protective and pathological roles of nitric oxide in endotoxin shock:Cardiovasc. Res. 1992; 26:48-57. Wu, C. C., Chen, S. J., Szabo, C., Thiermann, C., and Vane, J. R.:Aminoguanidine attenuates the delayed circulatory failure and improves survival in rodent models of endotoxic shock. Br. J. Pharmacol. 1995;114:1666-72. Wu, C. C., and Yen, M. H.:Beneficial effects of dantrolene on lipopolysaccharide-induced haemodynamic alterations in rats and mortality in mice. Eur. J. Pharmacol. 1997; 327:17-24.

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系統識別號 U0007-1704200714450507
論文名稱(中文) 在體外及體內試驗評估嵌段式共聚合物微膠體的傳遞基因效力
論文名稱(英文) Evaluation of the Block Copolymeric Micelle-Mediated Gene Transfer In Vitro And In Vivo
校院名稱 臺北醫學大學
系所名稱(中) 藥學研究所
系所名稱(英) Graduate Institute of Pharmacy
學年度 88
學期 2
出版年 89
研究生(中文) 蕭斐斤
學號 M8701010
學位類別 碩士
語文別 中文
口試日期
論文頁數 0頁
口試委員 指導教授-廖嘉鴻
關鍵字(中) 基因療法
聚合物
體外試驗
體內試驗
關鍵字(英) gene therapy
polymer
in vitro
in vivo
學科別分類
中文摘要 摘要 為了評估嵌段式共聚合物用於基因穿透上的可行性,故選用一個帶有經巨細胞病毒啟動子啟動的大腸桿菌基因 LacZ 為報告基因的質體 DNA。體外試驗乃利用在人類肝臟腫瘤 Hep G-2細胞株或小鼠腎上腺皮質的癌細胞株 Y-1上,加入經過不同比例混合的嵌段式共聚合物與 DNA,經過 X-gal 染色後,可比較出在 0.5-1% 的嵌段式共聚合物濃度範圍下可得到較佳的基因表現效果。除此之外,在 0.5-1% 濃度範圍下的細胞毒性經MTT分析後皆大於 IC50,且製劑的粒徑分布和界面電位分別在 160nm 和 —4.4mV。當額外給予細胞一些影響細胞內傳遞途徑的抑制劑時,發現報告基因表現量降低,因此推測傳遞途徑可能為 endocytosis。在動物實驗方面,在兔子及裸鼠眼睛局部滴入 DNA與嵌段式共聚合物的製劑後,可在兔子的虹膜及裸鼠眼球內部發現有來自報告基因的藍綠色表現,若投予可打開tight junction的試劑,在定量及定性實驗上,皆可發現就基因表現量和能出現報告基因表現的眼球組織面積皆呈增加的趨勢。故由這些結果,推測這種嵌段式共聚合物在體內及體外皆有可能成為一頗具潛力的傳遞基因的製劑。
英文摘要 Abstract In order to evaluate the feasibility of using the block copolymer for gene transfer, a plasmid (pCMV-??gal) carring the Escherichia coli ??galactosidase gene (LacZ) driven by a CMV promoter was used as a reporter. Different ratios of copolymer and plasmid DNA were prepared and added to human hepatocarcinoma Hep-G2 and mouse adrenal tumor Y-1 cell lines in vitro as well as by eye drops in ocular tissues of rabbits and mouse in vivo. In addition, the physical-chemical properties of polymeric micelles/plasmid polymeric micelles carrier, including Zeta potential, partical size, and critical micelle concentration (CMC) measurements were analyzed. The results have found that the block polymeric micelles were formed above 0.1 %(W/V) of block copolymer with 160 nm size and —4.4 mV potential. After staining with X-gal at 72 hours post-treatment, the most efficient reporter expression occurred when 0.5%-1% concentrations of the block copolymers were used as the carrier. In addition, the cytotoxicity for the Hep-G2 and Y-1 cells was determined by MTT assay and it was found that the IC50 of the block copolymer was 1% and 1.8% (IC50= 1, 1.8%), respectively. Furthermore, the reporter expression was detected around the iris of rabbits and the intraocular tissues of mice upon in vivo topically applying the copolymer/DNA formulationfor 48 hours. In the meantime, after some enhancer and endocytosis inhibotrs, the transport mechanisms of block copolymeric micelles was found probably through endocytosis in cultured cells and animals as well as enhancement through tight junction pathway. These in vitro and in vivo experiments postulate the possible potential usage of the block copolymers for DNA transfer.
論文目次 標題目錄 ABSTRACT4 摘要5 緒論:6 1-1基因療法(GENE THERAPY)6 1-2 關於傳遞基因載體的介紹6 1-2.1 病毒性載體7 1-2.1.1 Retrovirus7 1-2.2 Adenovirus8 1-2.3 Adeno-Associated Virus (AAV)9 1-2.4 Other viral vectors9 1-3 非病毒性載體9 1-3.1 Naked DNA10 1-3.2 Liposome10 1-3. 3. 陽離子性聚合物12 1-5 實驗目的12 實驗材料與方法:14 2-1 細胞培養 (CELL CULTURE):14 2-1.1 Hep G-2 細胞株14 2-1.2 Y-1細胞株14 2-2 質體的準備:14 2-2.1 質體14 2-2.2 轉型作用(Transformation)15 2-2.3 質體DNA的大量製備15 2-3 臨界微膠體濃度的測定16 2-4 製劑物理化學性質的測定16 2-4.1 粒徑大小的測定16 2-4.2 界面電位的測定16 2-5 細胞毒性的測定 - MTT ASSAY16 2-6 體外試驗17 2-6.1 DNA-聚合物微膠體製劑的配製17 2-6.2 DNA傳遞試驗17 2-6.3 關於DNA傳遞途徑的探討18 2-6.4 ?-gal 的定性分析18 2-6.5 ?-galactosidase 的活性定量分析19 2-7 體內試驗19 2-7.1雄性紐西蘭白兔(male New Zealand white rabbit)19 2-7.2雄性裸鼠(BALB/c-nu)21 結果與討論:23 3-1 嵌段式共聚合物對細胞生存率的影響23 3-2 臨界微膠體濃度的測定23 3-3 DNA-聚合物製劑的物理化學性質測定24 3-3.1 粒徑大小的測定24 3-3.2 界面電位的測定25 3-4 體外試驗25 3-4.1 不同的基因濃度對轉染效果造成的影響25 3-4.2 不同的聚合物濃度對轉染效果的影響26 3-4.3 製劑傳送機制的探討26 3-5 體內試驗28 3-5.1 ?-gal在裸鼠眼睛中的表現情形28 3-5.2 ?-gal在兔子眼睛中的表現情形28 3-6 製劑進入眼睛途徑的探討28 結論30 參考文獻45
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系統識別號 U0007-1704200714450626
論文名稱(中文) 天然物對胰島素及一氧化氮活性之調控
論文名稱(英文) Regulatory Activities of Natural Products on Insulin Secretion and Nitric Oxide Synthase.
校院名稱 臺北醫學大學
系所名稱(中) 生藥學研究所
系所名稱(英) Graduate Institute of Pharmacognosy
學年度 88
學期 2
出版年 89
研究生(中文) 林慧宜
學號 M8703005
學位類別 碩士
語文別 中文
口試日期
論文頁數 74頁
口試委員 指導教授-楊玲玲 教授
指導教授-許重輝 主任
關鍵字(中) HIT-T15 細胞株
RAW 264.7 細胞株
胰島素
一氧化氮合成脢
多醣體
香豆素
關鍵字(英) HIT-T15
RAW 264.7
Insulin
NOS
Polysaccharides
Coumarins
學科別分類
中文摘要 糖尿病為一種全身性慢性疾病,特別是其長久以來所導致之合併症如皮膚病變、發炎反應及敗血症都會對糖尿病患者造成危害。因此本論文中主要以天然之19種coumarins及28種polysaccharides進行其對誘導胰島素分泌及抗發炎之生理活性為目標而分為第一部份是天然物誘導胰島素分泌及作用機轉探討,第二部份是天然物對一氧化氮活性調控及消炎相關作用機制研究。 第一部份有關誘導Insulin分泌之研究,應用RIA 定量分析法測定20種coumarins及28種polysaccharides對胰臟b細胞(HIT-T15 cells) 誘導Insulin分泌,結果發現Coumarins之Phellopterin具有明顯誘導胰島素釋放作用,其對胰島素分泌機制受EDTA, EGTA, Nifedipin作用而分別降低33%, 60% 及92% 。因此可推斷Phellopterin誘導胰島素分泌作用之機轉與Ca+2有關。第二部份以開發NSAID(Non Steroid Anti-Inflammatory Drug)消炎藥物為目標;首先選用RAW 264.7 macrophage cells 以LPS誘導NO與PGE2產生,並分別以Griess reaction及PGE2 kit測定NO及PGE2含量。 在20種 Coumarins成份中Glabra-lactone能有效抑制NO及PGE2生成,其IC50分別為3.85 mg/mL與35.58 mg/mL,且以MTT分析細胞毒性試驗時Glabra-lactone並無顯著之毒性產生。以LPS誘導iNOS enzyme活性後Glabra-lactone並無法以直接或間接方式抑制iNOS enzyme活性。進一步以western blot分析 Glabra-lactone具有抑制iNOS及COX-2蛋白表現之作用,因而具有抑制NO自由基大量產生及消炎之作用。
英文摘要 Diabetes mellitus is a generalized chronic disease and the complication of diabetes mellitus including dermopathy, inflammation and sepsis occur frequently in patients. In this study, nineteen coumarins- related compounds and twenty-eight polysaccharides isolated from natural products were used to study their effects on insulin secretion and anti-inflammation in HIT-T15 and RAW 264.7 cells, respectively. In HIT-T15 cells, the effects of coumarins and polysaccharides extracts were studied on insulin release by radioimmuno assay (RIA). Among these courmarins, phellopterin obviously stimulated insulin secretion and the insulin secretion was decreased by 33%, 60% and 92% in EDTA, EGTA and Nifedipine added reaction. On the other experiment, glabra-lactone , a kind of courmarins showed the significant inhibitory activity on LPS-induced NO and PGE2 production in RAW 264.7 macrophage cells and the IC50 value is 3.85 mg/mL and 35.58 mg/mL, respectively. By MTT assay, glabra-lactone showed less cytotoxicity in RAW 264.7 cells. In the same experiment, glabra-lactone was unable to inhibit NOS enzyme activity by indirect or direct NOS enzyme activity assays. Western blot analysis of iNOS and COX-2 proteins, showed that glabra-lactone effectively inhibited the expression of the LPS-inducible iNOS and COX-2 genes. Therefore, glabra-lactone might be a promising natural product in the treatment of LPS-induced response and deserves further study.
論文目次 目錄考試委員名錄致謝 目錄 ……………………………………………………………... i 縮寫表 …………………………………………………………... v 中文摘要 ………………………………………………………... vi 英文摘要 ………………………………………………………... vii 緒論 ……………………………………………………………... viii 第一部份 天然物對胰島素分泌機制之調控 1 前言 ………………………………………………………….…. 2 實驗部分 ………………………………………………………. 7 I.實驗材料 ……………………………………………………. 7 I-1中藥材料及天然物 …………………………………….. 7 I-1-1天然物之多醣體 ……………………………….…. 7 I-1-2 Coumarins化合物及結構式 ……………………… 7 I-2細胞株 ………………………………………………….. 13 I-3細胞培養液 …………………………………………….. 13 I-4一般化學試藥及試劑…………………………………… 13 I-5套裝試劑組 …………………………………………….. 13 I-6儀器 …………………………………………………….. 14 II.實驗方法…………………………………………………….. 14 II-1多醣體之抽提方法 ……………………………………. 14 II-2藥品配製方法 …………………………………………. 14 II-3培養液配製方法 ………………………………………. 15 II-4一般溶液配製方法 ……………………………………. 15 II-5 HIT-T15 cells培養方法 ………………………………. 15 II-6 HIT-T15 cell 生長曲線之製作 ………………………. 15 II-7 HIT-T15 cells誘導胰島素之生物活性檢測 …………. 16 II-8胰島素定量免疫分析方法 ……………………………. 16 II-9 NO含量測定 ………………………………………….. 16 II-10統計資料 ………………………………………….….. 17 結果 …………………………………………………………….. 18 1. HIT-T15細胞生長曲線 ………………………………… 18 2. 胰島素放射免疫分析稀釋試驗 ………………………… 19 3. 葡萄糖時間依存分析劑量依賴分析…………………….. 20 4. 多醣體誘導胰島素之生物活性檢測 …………………… 22 5. Coumarins誘導胰島素之生物活性檢測 ………………... 25 6. Phellopterin對胰島素及NO之時間依存分析 …………. 28 7. Phellopterin對胰島素及NO之劑量依賴分析 ………….. 30 8. 作用劑與Phellopterin對胰島素及NO含量分析 ……… 32 9. 天然物對胰島素及NO含量分析 ……………………… 35 討論 …………………………………………………………….. 37 第二部份 天然物對一氧化氮活性之調控 43 前言……………………………………………………………… 44 實驗部分 ………………………………………………………. 47 I. 實驗材料 …………………………………………………... 47 I-1藥材 …………………………………………………….. 47 I-2細胞株 ………………………………………………….. 47 I-3培養液 ………………………………………………….. 47 I-4一般化學試藥 ………………………………………….. 47 I-5免疫試劑………………………………………………… 49 I-6套裝試劑組 …………………………………………….. 49 I-7儀器 …………………………………………………….. 49 II實驗方法 …………………………………………………… 49 II-1. RAW 264.7 cell 培養液之調製方法 ………………… 49 II-2藥品之配製方法………………………………………... 49 II-3一般溶液之配製方法 ………………………………… 50 II-4 應用MTT assay測定細胞毒性 ……………………… 52 II-5 NO含量測試 ………………………………………….. 53 II-6 iNOS enzyme activity 測試 …………………………... 53 II-7 Western blot分析法 …………………………………… 54 II-8 PGE2 assay ……………………………………………... 54 II-9 HIT-T15及RAW264.7 cells co-culture 實驗 ………... 55 結果 …………………………………………………………….. 56 1. Coumarins抑制NO含量分析 ……………………….….. 56 2. Coumarins抑制NO之IC50及細胞毒性分析 ………….. 59 3. iNOS 酵素活性測試 ……………………………………. 60 4. Glabra-lactone抑制NO時間依存及劑量依賴分析 ….…. 61 5. Glabra-lactone抑制iNOS,COX-2之western blot 分析法. 63 6. PGE2含量測定 ………………………………………….. 64 7. HIT-T15及RAW 264.7 cells co-culture試驗…………….. 65 討論 …………………………………………………………….. 66 參考文獻 ………………………………………………………. 68
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系統識別號 U0007-1704200714450895
論文名稱(中文) 生物可降解性牙用/骨用聚乳酸高分子摻合物的製備與鑑定
論文名稱(英文) Preparation and Characterization of Biodegradable PLA Polymeric Blends for Dental and Orthopedic application
校院名稱 臺北醫學大學
系所名稱(中) 牙醫學系碩博士班
系所名稱(英) Graduate School of Dentistry
學年度 89
學期 2
出版年 90
研究生(中文) 闕如玉
學號 m8804014
學位類別 碩士
語文別 中文
口試日期
論文頁數 100頁
口試委員 指導教授-李勝揚
指導教授-陳建中
關鍵字(中) 生物可吸收性
摻合
界面活性劑
聚左乳酸
聚左右乳酸
關鍵字(英) Bioresorbable
Blend
Surfactant
Poly- L-lactic acid
Poly- DL-lactic acid
學科別分類
中文摘要 本研究目的是進行體內可吸收性高分子複合材料的改質,以期更適於口腔顎面外科、整形外科及骨科之應用。以二氯甲烷為共同的溶劑,分別溶解基質-聚左乳酸(PLLA)、第二添加物-聚左右乳酸(PDLLA)或聚己內酯(PCL)及界面活性劑-聚乙烯氧化聚合物-聚丙烯氧化聚合物(PEOPPO),將此三種成分進行不同比例的摻合,其中PLLA和PDLLA的重量百分比為100/0、80/20、60/40、50/50、40/60、20/80及0/100。實驗上利用熱重分析儀、微差掃描式熱分析儀(DSC)、紅外線光譜測定儀、膠體滲透層析儀、掃描式電子顯微鏡及材料機械性質測定儀來量測結晶熔點、玻璃轉化溫度、相的表現及機械特性。由DSC結果發現,沒有添加界面劑之PLLA/PDLLA摻合物有兩個玻璃轉化溫度的趨勢,而在添加界面劑後的摻合物其玻璃轉化溫度會隨著PLLA成分的減少而降低,且呈線性的轉移。在機械性質的比較上,未添加界面活性劑前PLLA/PDLLA摻合物最佳強度的摻合比例為40/60,而適當的界面活性劑濃度為2%,此時PLLA/PDLLA摻合物最佳強度的摻合比例為50/50。PLLA添加PCL後,伸長量(elongation)增加最多,但強度變弱。觀察摻合物斷面型態,發現硬而韌的材料具有絲絮狀不光滑之斷面。綜合本研究的結果發現,以摻合的方式加入界面活性劑進行高分子材料的改質可找到一最佳的組成分且具較優的機械性質。
英文摘要 The purpose of this study is to improve the properties of bioresorbable materials for particular applications in dental and orthopedic surgery. Blends of biodegradable poly-L-lactic acid (PLLA), poly- DL-lactic acid (PDLLA) or polycaprolactone (PCL) , and a third component, the surfactant PEO-PPO, were prepared by blending these three polymers at various ratios using dichloromethane as a solvent. The weight percentages of PLLA/PDLLA (or PCL) blends were 100/0, 80/20, 60/40, 50/50, 40/60, 20/80 and 0/100, respectively. Physical and morphological properties such as crystalline melting point, glass transition point, phase behaviors, degradation behavior and mechanical properties were characterized by thermogravimetric analysis, differential scanning calorimetry, infrared spectroscopy, gel permeation chromatography, scanning electron microscopy and dynamic mechanical analysis. DSC data indicate that PLLA/PDLLA blends without PEOPPO have two Tgs. With the addition of PEOPPO, there is a linear shifting of Tg as a function of composition showing a lesser percentage of PLLA, and a lower glass transition temperature indicating better miscibility has been achieved. DMA data show the best composition of PLLA/PDLLA blends without PEOPPO is 40/60, and the best concentration of PEOPPO is 2%. The 50/50 PLLA/PDLLA/2%PEOPPO blend has better mechanical properties. Elongation of PLLA increases while adding PCL, but the strength decreases at the same time. SEM observation shows that harder and tougher materials have rougher fracture surface morphology.
論文目次 中文摘要..............................................I 英文摘要..............................................III 目錄..................................................V 圖表目錄..............................................VI 第一章緒論.........................................1 1.1 前言..............................................1 1.2 研究目的與動機....................................3 第二章 文獻回顧.......................................5 2.1 聚乳酸............................................7 2.2 聚己內酯..........................................10 2.3 聚乙烯氧化聚合物-聚丙烯氧化聚合物.................11 2.4 摻合..............................................12 第三章 研究材料與方法................................14 3.1 材料與試劑........................................15 3.2 儀器設備..........................................15 3.3 研究方法與進行步驟................................16 3.3.1聚左乳酸/聚左右乳酸或聚己內酯/聚乙烯氧化聚合物-聚丙烯氧化聚合物摻合物的製備......................................16 3.3.2 摻合物測試......................................16 3.3.2.1 GPC測試.......................................16 3.3.2.2 FT-IR測試.....................................16 3.3.2.3 X-ray測試.....................................17 3.3.2.4 TGA測試.......................................17 3.3.2.5 DSC測試.......................................17 3.3.2.6 DMA測試.......................................17 3.3.2.7 SEM觀察.......................................18 3.3.3 統計............................................18 第四章 結果與討論....................................19 4.1 FT-IR測試.........................................19 4.2 GPC測試...........................................19 4.3 X-ray測試.........................................20 4.4 DSC測試...........................................21 4.5 DMA測試...........................................23 第五章 結論..........................................29 參考文獻..............................................30 Table 1Wide Angle X-ray diffraction of PLLA/PDLLA and PLLA/PDLLA/2%PEOPPO blends.......................35 Table 2Wide Angle X-ray diffraction of PLLA/PCL and PLLA/PCL/2%PEOPPO blends ..........................36 Table 3Thermal onset degradation temperature of PLLA/PDLLA and PLLA/PDLLA/PEOPPO blends...................... 37 Table 4Thermal onset degradation temperature of PLLA/PCL/PEOPPO blends.............................38 Table 5DSC data of PLLA/PDLLA blends (second heating).....39 Table 6DSC data of PLLA/PDLLA/PEOPPO blends (second heating) ..................40 Table 7DSC data of PLLA/PCL blends (second heating).......42 Table 8DSC data of PLLA/PCL/PEOPPO blends (second heating).43 Table 9Mechanical properties of PLLA/PDLLA blends..........46 Table 10Mechanical properties of 50/50 PLLA/PDLLA blends with different concentration PEOPPO...............................47 Table 11Mechanical properties of PLLA/PDLLA /PEOPPO blends....................................................48 Table 12Mechanical properties of PLLA/PCL blends............50 Table 13 aMechanical properties of PLLA/PCL/0.5%PEOPPOblends..51 Table 13 bMechanical properties of PLLA/PCL/1%PEOPPO blends...52 Table 13 cMechanical properties of PLLA/PCL/2%PEOPPO blends...53 Table 14Dynamic mechanical properties of PLLA/PDLLA blends..54 Figure 1Formula of PLLA.....................................55 Figure 2Formula of PCL......................................56 Figure 3Formula of PEOPPO...................................57 Figure 4(A)Lactate converses to pyruvate....................58 (B) The metabolism pathway of pyruvate .............59 Figure 5FT-IR spectrum of PLLA/PDLLA blends...............60 Figure 6FT-IR spectrum of PLLA/PCL blends...................61 Figure 7GPC data of PLLA/PDLLA blends.......................62 Figure 8GPC data of PLLA/PDLLA/2%PEOPPO blends..............63 Figure 9GPC data of PLLA/PCL blends.........................64 Figure 10GPC data of PLLA/PCL/2%PEOPPO blends................65 Figure 11Wide Angle X-ray diffraction pattern of PLLA/PDLLA blends..............................................66 Figure 12Wide Angle X-ray diffraction pattern of PLLA/PDLLA /PEOPPO blends...........................67 Figure 13Wide Angle X-ray diffraction pattern of PLLA/PCL blends..............................................68 Figure 14Wide Angle X-ray diffraction pattern of PLLA/PCL/PEOPPO blends..............................69 Figure 15Initial degradation temperature of PLLA/PDLLA and PLLA/PDLLA/PEOPPO blends............................70 Figure 16DSC data of PLLA/PDLLA blends (2nd heating )........71 Figure 17(A)Tg of PLLA/PDLLA blends (2nd heating )...........72 (B)△Hm of PLLA/PDLLA blends (2nd heating )........73 Figure 18DSC data of PLLA/PDLLA/0.5%PEOPPO blends............74 Figure 19DSC data of PLLA/PDLLA/2%PEOPPO blends..............75 Figure 20Tg of PLLA/PDLLA/PEOPPO blends (2nd heating ).......76 Figure 21Tcc of PLLA/PDLLA/PEOPPO blends (2nd heating )......77 Figure 22△Hcc of PLLA/PDLLA/PEOPPO blends (2nd heating )....78 Figure 23Tm of PLLA/PDLLA/PEOPPO blends (2nd heating ).......79 Figure 24△Hm of PLLA/PDLLA/PEOPPO blends (2nd heating ).....80 Figure 25DSC data of PLLA/PCL blends (2nd heating )..........81 Figure 26(A)Tm of PLLA/PCL blends (2nd heating ) ............82 (B)△Hm of PLLA/PCL blends (2nd heating )..........83 Figure 27DSC data of PLLA/PCL/0.5%PEOPPO blends..............84 Figure 28DSC data of PLLA/PCL/1%PEOPPO blends................85 Figure 29DSC data of PLLA/PCL/2%PEOPPO blends ...............86 Figure 30Tcc of PLLA/PCL/PEOPPO blends (2nd heating )........87 Figure 31△Hcc of PLLA/PCL/PEOPPO blends (2nd heating )......88 Figure 32△Hm of PLLA/PCL/PEOPPO blends (2nd heating ).......89 Figure 33SEM of PLLA×500 (surface)..........................90 Figure 34SEM of PLLA×1.5k (surface).........................91 Figure 35SEM of PDLLA×500 (surface).........................92 Figure 36SEM of PCL×500 (surface)...........................93 Figure 37SEM of 50/50 PLLA/PDLLA/0.5%PEOPPO (fracture).......94 Figure 38SEM of 50/50 PLLA/PDLLA/2%PEOPPO (fracture).........95 Figure 39SEM of 80/20 PLLA/PDLLA/2%PEOPPO (fracture).........96 Figure 40SEM of 80/20 PLLA/PCL blend (fracture)..............97 Figure 41SEM of 50/50 PLLA/PCL blend (fracture)..............98 Figure 42SEM of 100/0 PLLA/PCL/0.5%PEOPPO blend (fracture)...99 Figure 43SEM of 80/20 PLLA/PCL/0.5%PEOPPO blend (fracture)..100
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