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系統識別號 U0007-0308201111314500
論文名稱(中文) 血小板濃厚血漿釋放生長因子的特性評估及其在攝護腺根除手術後勃起功能恢復的研究
論文名稱(英文) Characterization of platelet-rich plasma releasing growth factors and its application on recovery of erectile function during radical prostatectomy
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 99
學期 2
出版年 100
研究生(中文) 吳宜娜
研究生(英文) Yi-No Wu
學號 M301098015
學位類別 碩士
語文別 中文
口試日期 2011-07-19
論文頁數 99頁
口試委員 委員-蔡義弘
委員-林山陽
委員-江漢聲
委員-許明照
指導教授-何秀娥
中文關鍵字 陰莖勃起功能障礙  生長因子  血小板濃厚血漿  攝護腺根除手術 
英文關鍵字 erectile dysfunction  growth factor  platelet-rich plasma  radical prostatectomy 
學科別分類
中文摘要 攝護腺根除手術是侷限性攝護腺癌病患最佳的手術治療方式,然而進行攝護腺切除手術後,陰莖勃起功能障礙至今仍是患者最主要的併發症。血小板濃厚血漿含有許多自體的血小板生長因子,並被廣泛應用於再生醫學。所以本研究的目的主要探討血小板濃厚血漿釋放生長因子的生物特性並以神經損傷動物模式評估其對勃起功能恢復之效果。在本實驗中以酵素免疫吸附分析法 (ELISA) 定量血小板衍生生長因子 AB (PDGF-AB) 來評估抗凝血劑種類、活化因子種類及其濃度、溫度、作用時間與氯化鈣 (CaCl2) 影響血小板釋放生長因子的效果,並藉此建立最佳化的血小板濃厚血漿製備方法。結果顯示使用Anticoagulant Citrate Dextrose Solution A (ACD-A) 抗凝血劑製備血小板濃厚血漿後再以幾丁聚醣 (chitosan) 活化儲存在 -20℃、15天,相較於其他條件可以獲得較多的生長因子釋放。
接著我們建立海綿體神經損傷動物模式,將 18 隻 12 周齡的大鼠分為三組:一組進行假手術,一組進行損傷控制組,最後一組進行神經損傷後血小板濃厚血漿的治療。四週後,結果顯示使用血小板濃厚血漿治療的大鼠,海綿體內壓力相較於損傷控制組為高。組織染色結果顯示以血小板濃厚血漿治療,可以降低陰莖海綿體組織纖維化,同時保留較多的軸突髓鞘。基因表現結果顯示使用血小板濃厚血漿治療後亦可以降低 TGF-ß1 的表現量。這些結果顯示將血小板濃厚血漿應用於海綿體組織上可以促進勃起功能的恢復,在海綿體神經也有潛在的修復效果,可以提供血小板濃厚血漿未來在臨床上新的應用。
英文摘要 Radical prostatectomy (RP) is the best method of surgical therapy for organ-confined prostate cancer patients. However, even now, erectile dysfunction is a major complication after undergoing RP. Platelet rich plasma (PRP) contains autologous thrombocyte growth factors and has been applied in regenerative medicine. The aim of this study was to investigate the characterization of PRP releasing growth factors and the effects of PRP in restoring of erectile function in a nerve injury rat model. Preliminarily, the effects of various anticoagulants, activating factors, reaction temperature, time and calcium chloride on the growth factors release after human platelets activation were examined to optimize the method of PRP preparation. The platelet-derived growth factor AB (PDGF-AB) releasing from PRP was assayed by ELISA. Results demonstrated that greater concentrations of growth factors were released in PRP when it was prepared with ACD-A as anticoagulant and stimulated with chitosan incubated at -20℃ for 15 days than after the other conditions.
Subsequently, we established a cavernous nerve (CN) injury rat model and 12-week-old Sprague-Dawley male rats (n=18) were randomly divided into three equal groups: (1) sham operation; (2) vehicle only group: underwent bilateral nerve crushing without treatment; (3) PRP group: underwent bilateral nerve crushing with an immediate treatment of PRP on the corpus cavernosum. Four weeks after surgery, in the group that underwent bilateral nerve crush and then treated with normal saline, the functional evaluation showed a lower mean intracavernous pressure (ICP) than that in the sham group. PRP treatments resulted in significant recovery of erectile function, as compared with vehicle only group. Histologically, the group with the treatment of PRP had significantly less fibrosis of corpus cavernosum and a significant preservation of myelinated axons of CNs compared with vehicle only group. mRNA expression of TGF-ß1 in corpus cavernosum tissue was found to significantly decrease in PRP group compared with vehicle only group. It was concluded that the application of PRP to the site of corpus cavernosum after RP facilitates recovery of erectile function. Our research indicates that PRP has potentials of being clinically applicable to restore CN function during RP.
論文目次 目錄 I
附表目錄 IV
附圖目錄 V
中文摘要 VIII
ABSTRACT X
壹、 緒論 1
第一節 研究背景介紹 1
一、攝護腺癌 (Prostate cancer) 1
(一)攝護腺癌發生率與死亡率 1
(二)攝護腺癌的症狀 2
(三)攝護腺癌診斷 3
(四)攝護腺癌的分級與分期 5
(五)攝護腺癌治療及其併發症 8
二、海綿體神經損傷的動物模式 (Animal models of cavernous nerve (CN) injury) 9
(一)海綿體神經概論 9
(二)海綿體神經損傷的動物模式的發展 10
(三)生長因子在海綿體神經損傷後的調節作用 14
三、血小板 (Platelet) 17
(一)血小板的組成 17
(二)血小板活化機轉 17
(三)血小板中主要生長因子的生物功能 22
四、血小板濃厚血漿 (Platelet-rich plasma, PRP) 25
(一)血小板濃厚血漿定義 25
(二)血小板濃厚血漿製備 25
(三)血小板濃厚血漿在醫學上的應用 25
(四)血小板濃厚血漿未來展望 28
第二節 研究動機與目的 28
貳、 實驗材料與方法 30
第一節 實驗材料 30
一、抽血樣本 30
二、試劑藥品 30
三、耗材 32
四、套裝實驗組 33
第二節 儀器設備 33
第三節 實驗方法 34
ㄧ、PRP 的製備及其特性評估 34
(一)PRP 的製備 34
(二)影響PRP釋放生長因子的特性評估 35
(三)酵素免疫吸附分析 (Enzyme-linked immunosorbent assay) 39
二、建構雙側海綿體神經損傷的動物模式 39
(一)實驗動物 39
(二)雙側海綿體神經損傷與陰莖勃起功能測定 40
三、PRP 對陰莖勃起功能恢復效果之評估 41
(一)陰莖勃起功能評估 41
(二)組織學評估 41
(三)基因表現評估 45
(四)分析與統計 47
叁、 結果與討論 48
第一節 PRP 的製備及其特性評估 48
ㄧ、抗凝血劑種類對於 PRP 釋放生長因子的影響 48
二、活化因子對於PRP釋放生長因子的影響 51
(一)活化因子種類及其濃度對於 PRP 釋放生長因子的影響 51
(二)活化因子種類及其作用時間對於 PRP 釋放生長因子的影響 59
三、溫度對於 PRP 釋放生長因子的影響 65
四、活化因子合併溫度對於 PRP 釋放生長因子的影響 68
五、CaCl2 對於 PRP 釋放生長因子的影響 72
第二節 建構雙側海綿體神經損傷的動物模式 75
第三節 PRP 對陰莖勃起功能恢復效果之評估 77
一、陰莖勃起功能評估 77
二、組織學評估 77
(一)海綿體神經與陰莖背神經的型態結構變化評估 77
(二)陰莖海綿體中第一型膠原蛋白與第三型膠原蛋白的變化評估 82
(三)海綿體神經富含髓鞘軸突的數量與陰莖背神經中髓鞘的型態變化評估 82
(四)陰莖海綿體中細胞凋亡的數量評估 83
三、基因表現評估 87
肆、 結論 90
伍、 參考文獻 91

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