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Proteomic Analysis of Experimentally Induced Stress Urinary Incontinence in Mice

By 林雨寗 and Yu-Ning Lin

Abstract

尿失禁 (Incontinence) 不是一種致命性疾病,但是卻影響了世界上八分之一女性的生活品質,該病限制了這些患者的日常社交生活,造成情緒困擾和社會隔離感,可能進而影響身體健康。國際尿控協會 (International Continence Society,ICS)瞭解到這是個不容小覷的疾病,在1979年時將尿失禁定義為「任何尿液非自主性的漏出現象」,對於尿失禁的診斷或治療等,有了較明確的規範。而在尿失禁的分類中,又以應力性尿失禁 (Stress Urinary incontinence,SUI) 最為常見,幾乎每三位婦女就有一位曾經經歷應力性尿失禁,它的成因多與外因性禁尿機制缺損有關,例如懷孕、生產、骨盆底肌鬆弛或尿道括約肌閉鎖功能不全。其中又以懷孕對骨盆神經及肌肉的傷害扮演最常見的角色。然而,應力性尿失禁的罹病率如此高,就醫率卻很低,也因此對於該病的機制仍不清楚,遂以本論文探討其中相關機制,希望能為有困擾的患者尋求解決之道。 在本實驗中利用擴張小鼠陰道來模擬產傷,然後再從蛋白質體學的角度去探討小鼠尿道括約肌在模擬婦女生產過程對應力性尿失禁的生理意義。實驗採用18隻六~八週大的C57BL/6 母鼠,分成陰道擴張0 mm (control)、3 mm (sham) 和8 mm(treat) 三組, 在擴張陰道兩天後進行小鼠膀胱造管手術 (Suprapubic Tube Implantation,STI),手術後兩天在麻醉中測量小鼠漏尿點壓力 (Leaking Point Pressure,LPP),結束後隨即犧牲小鼠,取其尿道組織做螢光二維膠體電泳分析(Two-Dimensional Difference gel Electrophoresis,DIGE)、切片染色及西方墨點法(Western blot)。 結果顯示,陰道擴張後的漏尿點壓力在control、sham 和treat 組分別為48.5、21.3 和8.78 cmH2O,sham 和treat 組較於control 組皆有顯著差異,平均偏低了39.7 cmH2O。在螢光二維膠體電泳分析則鑑定到68 個差異性表現的蛋白質點,這些蛋白質所參與的路徑可能決定了尿失禁發展的關鍵,其中myosin 在treat 組較於control 組少了2.7 倍, 與本實驗室研究方向相近, 在western blot 、 immunohistochemistry 和immunoflourence 我們也證明treat 組只有control 組的0.24倍、0.2 倍和0.23 倍,於是我們推測myosin 為應力性尿失禁之關鍵,希望此研究可以幫助釐清應力性尿失禁的成因,使尿失禁病患可以免於手術之苦。Urinary incontinence (UI) is not a fatal disease, but it negatively affects the quality of life of millions of women. It causes emotional distress and social disassociation. Stress urinary incontinence (SUI) is the most common type among UI as one of every three women ever experienced SUI. However, most women do not seek medical attention for this condition. Due to the low attendance rates of SUI patients, the pathogenesis of SUI remains unclear. This study was conducted to evaluate protein alterations in the urethras of mice with experimentally induced SUI. In this study, we dilated the vagina of mice to simulate birth trauma for inducing SUI.Eighteen female C57BL/6 mice were randomly distributed into three groups. Two groups underwent vaginal distention (VD) for 1 hour with 3 mm (sham) and 8 mm (treat) dilators each, and the others would be the control group. Two days after VD, all mice underwent suprapubic tube implantation (STI), and the following two days processed leak-point pressure (LPP) measurement. After LPP measurement, all mice were sacrificed and harvested their urethras for western blot, two-dimensional difference gel electrophoresis (DIGE), masson stain, immunoflourence and immunohistochemistry. The result showed that the LPP was significantly decreased in the 3 mm and 8 mm VD groups compared with that in the control group (48.5, 21.3 and 8.78 cmH2O, respectively), and showed 68 differentially expressed protein spots in 2D-DIGE analysis. Masson stain showed no significant difference in urethra tissue among the three groups. The expression levels of myosin were 2.7 folds lower in treat group than in control group. The same trend was approved by immunoflourence, western blot and immunohistochemistry. Therefore, we assume that myosin plays an important role in SUI. We aim to elucidate the pathology of SUI and help the SUI patients free from surgery.致謝 ................................................................................................................................. I 中文摘要 ........................................................................................................................ II 英文摘要 ...................................................................................................................... III 目次 .............................................................................................................................. IV 表目次 .......................................................................................................................... VI 圖目次 .......................................................................................................................... VI 英文縮寫檢索表 ......................................................................................................... VII 第一章 緒言 ................................................................................................................ 1 第一節 文獻回顧 .................................................................................................. 1 一、人體膀胱與泌尿細胞的生理作用 ........................................................ 1 二、尿失禁的流行病學與診斷 .................................................................... 3 三、產傷對應力性尿失禁之影響 ................................................................ 5 四、雌激素對應力性尿失禁之影響 ............................................................ 6 五、應力性尿失禁之現有治療 .................................................................... 7 六、蛋白質體學探討 .................................................................................... 8 第二節 研究動機與策略 .................................................................................... 11 第二章 材料與方法 .................................................................................................. 13 第一節 應力性尿失禁動物模式建立 ................................................................ 13 一、實驗動物資訊 ...................................................................................... 13 二、陰道擴張 .............................................................................................. 13 三、造管手術 .............................................................................................. 13 第二節 應力性尿失禁生理功能測試 ................................................................ 16 一、漏尿點壓力測試 .................................................................................. 16 二、組織學觀察 .......................................................................................... 16 第三節 蛋白質體研究分析 ................................................................................ 17 一、蛋白質萃取 .......................................................................................... 17 二、蛋白質定量 .......................................................................................... 17 三、西方墨點法蛋白質分析 ...................................................................... 17 四、蛋白質螢光標定 .................................................................................. 18 五、二維螢光膠體電泳 .............................................................................. 20 六、銀染法 .................................................................................................. 20 七、比對軟體分析 ...................................................................................... 21 八、酵素水解膠體內蛋白質 ...................................................................... 21 九、質譜分析 .............................................................................................. 21 十、統計與路徑分析 .................................................................................. 21 十一、免疫組織染色 .................................................................................. 21 十二、免疫螢光染色 .................................................................................. 22 第三章 結果 .............................................................................................................. 23 第一節 小鼠陰道擴張前後之尿失禁生理分析 ................................................ 23 一、漏尿點壓力測試 .................................................................................. 23 二、組織染色切片分析尿道肌肉變化 ...................................................... 23 第二節 小鼠陰道擴張前後之蛋白質體分析 .................................................... 26 一、二維螢光膠體電泳分析與膠體銀染結果 .......................................... 26 二、比對軟體分析 ...................................................................................... 28 三、質譜分析及蛋白質資料庫與功能比對 .............................................. 28 四、路徑分析結果 ...................................................................................... 35 五、免疫組織染色證實Myosin在陰道擴張後的小鼠尿道有減少趨勢.. 38 六、西方墨點法證實Myosin在陰道擴張後的小鼠尿道有減少趨勢 .... 39 七、免疫螢光法證實Myosin在陰道擴張後的小鼠尿道有減少趨勢 .... 40 第四章 討論 .............................................................................................................. 41 第一節 小鼠陰道擴張與否之生理現象與蛋白質體探討 ................................ 41 第二節 針對myosin藥物開發治療尿失禁之探討 .......................................... 44 第五章 結論 .............................................................................................................. 46 第六章 未來計畫 ...................................................................................................... 47 第七章 參考文獻 ...................................................................................................... 48 附錄一、失禁與否的尿道基因變化............................................................................. 53 附錄二、尿失禁與否患者的尿道微陣分析................................................................. 55 附錄三、陰道擴張對應力性尿失禁之蛋白質點分析biological process................... 56 附錄四、陰道擴張對應力性尿失禁之蛋白質點分析 molecular function................. 59 附錄五、陰道擴張對應力性尿失禁之蛋白質點分析cell component........................ 60 附錄六、文獻發表 ....................................................................................................... 62 表目次 表1. 尿失禁的分類與常見原因 ................................................................................... 4 表2. 比對軟體分析小鼠陰道擴張與否對尿道蛋白之二維膠體電泳圖結果 ......... 29 圖目次 圖1. 膀胱生理示意圖 ................................................................................................... 2 圖2. 雌激素分泌正常與缺乏對尿失禁影響示意圖 ................................................... 6圖3. 質譜分析的組成元件........................................................................................... 10圖4. 實驗性誘導小鼠應力性尿失禁之實驗流程圖................................................... 12圖5. 陰道擴張器與擴張後兩天的小鼠陰道............................................................... 14 圖6. 膀胱造管手術 ..................................................................................................... 15圖7. 二維螢光膠體電泳流程圖................................................................................... 18圖8. 二維膠體電泳有無內校之比較圖....................................................................... 19圖9. 小鼠陰道擴張與否之漏尿點壓力測試圖........................................................... 24圖10. 小鼠陰道擴張實驗之中段尿道橫切面染色觀察............................................. 25圖11. 小鼠陰道擴張實驗之二維螢光膠體電泳圖..................................................... 26 圖12. 小鼠陰道擴張螢光二維膠體電泳銀染與取點圖............................................. 27圖13. 小鼠陰道擴張與否尿道蛋白質體分析............................................................. 34圖14. Myosin調節平滑肌收縮機制圖......................................................................... 35 圖15. Myosin序列比對分析結果................................................................................. 36 圖16. Myosin於DeCyder軟體分析圖......................................................................... 37 圖17. Myosin在小鼠尿道之免疫組織染色圖............................................................. 38 圖18. Myosin在小鼠尿道之西方墨點圖..................................................................... 39 圖19. Myosin在小鼠尿道之免疫螢光染色圖............................................................. 40 圖20. Myosin結構示意圖............................................................................................. 41 圖21. 肌肉收縮機制圖................................................................................................. 4

Topics: 應力性尿失禁, 漏尿點壓力, 肌凝蛋白, stress urinary incontinence, leak point pressure, myosin.
Publisher: 生命科學院碩士在職專班
Year: 2014
OAI identifier: oai:ir.lib.nchu.edu.tw:11455/81144
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