[[alternative]]Protein distribution in two-dimensional polyacrylamide gel electrophoresis induced by polypyridyl ruthenium complexes

碩士[[abstract]]抗癌藥物釕金屬錯合物在臨床醫學上對治療腫瘤上具有相當的發展潛力。基於其毒性低、水溶性高及有高度特異選擇性等特點，可比美抗癌藥物鉑金屬錯合物 (cisplatin)。本實驗是利用吡啶釕金屬錯合物會與DNA的鳥糞嘌呤 (guanine)的N7位置鍵結，進而影響DNA的複製及轉錄作用的進行的特性，近一步將不同結構的吡啶釕金屬錯合物加入大腸桿菌的培養中，結果發現吡啶釕金屬錯合物能有效抑制大腸桿菌的生長。萃取出大腸桿菌的蛋白質後，利用蛋白質的等電點及分子量不同將萃取出的蛋白質以等電焦集電泳 (IEF)及SDS-PAGE進行分離，再利用影像比對軟體2-D ImageMaster進行二維凝膠比對，結果發現加入吡啶釕金屬錯合物培養的大腸桿菌在蛋白質表現上的確與未加入吡啶釕金屬錯合物的控制組有所不同，總共發現有43個點有明顯變化。因此，將這43個變化的點切割下來，以胰蛋白酶(trypsin)將蛋白質消化成小片段胜肽，再以奈流液相層析質譜儀(nano LC-MS/MS)進行蛋白質的身分鑑定，進一步分析吡啶釕金屬錯合物所影響的生物功能及代謝路徑。結果發現被吡啶釕金屬所影響的蛋白質表現，多數是被抑制的，少數表現量反而是增多的，這些表現量增多的蛋白質多是能抵抗環境壓力的蛋白質，顯示大腸桿菌的生理功能及代謝路徑被改變，在多數蛋白質被抑制的情形下，可能使大腸桿菌無法執行正常的生理功能及代謝路徑，而導致菌體的死亡。本實驗證實了吡啶釕金屬不但可以抑制大腸桿菌的生長，更會影響大腸桿菌的總體蛋白質表現。藉由了解吡啶釕金屬錯合物所影響蛋白質的代謝路徑及所執行的生理功能，期望能找出吡啶釕金屬錯合物作用在生物體的可能機制，以提供未來設計及發展新藥物的方向。[[abstract]]Ruthenium complexes have the properties of low systemic toxicity, high water solubility, specificity to antimetastatic tumor, and the potential to displace the anticancer drug of cisplatin. It is interesting to explore the correlation between the structures of ruthenium complexes and the expression of protein. Polypyridyl ruthenium complexes were used to examine this concept. Polypyridyl ruthenium complexes are liable to bind with DNA in N7 position of guanine and lead to the termination of the DNA duplication and transcription. The cell growth was inhibited by using polypyridyl ruthenium complexes. In order to understand the correlation between cell inhibition and protein expression, proteomics was used. The variation of proteins distribution was accomplished by the extraction of E. coli totaprotein with different concentration of polypyridyl ruthenium complexes, the separation of protein in their different pI values and molecular weight by two-dimensional polyacrylamide gel electrophoresis, the analysis by 2-D ImageMaster, the digestion with trypsin, and the detection of the molecular ions with nano LC-MS/MS. The result showed that there were 43 spots altered conspicuously. Most protein expression were decreased, and few protein expression were increased. The functions of increased protein expression were the protein which could resist the stress of environment. It revealed that the physical function and metabolic pathway of E. coli was changed, and this reason may cause the death of cell. According to the result, the mechanism of polypyridyl ruthenium complexes acting on the cell may provide a consideration in ruthenium-base anticancer drug.[[tableofcontents]]謝誌.....................................................Ⅰ 中文摘要.................................................Ⅱ 英文摘要.................................................Ⅳ 目錄.....................................................Ⅴ 圖表目錄.................................................Ⅶ 符號與縮寫表.............................................Ⅹ 前言......................................................1 第一章 緒論 1-1吡啶釕金屬錯合物結構與細胞表現的關係.................3 1-1-1金屬錯合物在生物體內扮演的角色....................3 1-1-2金屬在醫學上的應用................................5 1-1-3抗癌藥物鉑金屬錯合物之簡介........................7 1-1-4釕金屬錯合物在生物體內的作用機制..................8 1-2蛋白質體學簡介......................................15 1-2-1何謂蛋白質體學...................................15 1-2-2蛋白質體學在標的藥物開發上的應用.................17 1-2-3蛋白質體學應用於微生物生理之研究.................20 1-2-4蛋白質體學的研究方法.............................21 1-2-5二維凝膠電泳在蛋白質體研究之應用.................23 第二章 材料與方法 2-1材料................................................28 2-1-1 E. coli BL21(DE3)..............................28 2-1-2 儀器設備........................................28 2-1-3 藥品............................................29 2-1-4試劑製備.........................................30 2-2實驗方法與步驟......................................35 第三章 結果與討論 3-1吡啶釕金屬錯合物對大腸桿菌生長之抑制................48 3-2大腸桿菌作用的蛋白質二維電泳圖分佈之探討............49 3-2-1[Ru(terpy)(bpy)Cl]Cl所造成的影響.................49 3-2-2[Ru(terpy)(bpy)Cl]Cl濃度變化所造成的影響.........51 3-2-3 Ru(bpy)2Cl2所造成的影響.........................56 3-2-4 Ru(bpy)2Cl2濃度變化所造成的影響.................57 3-2-5[Ru(terpy)(bpy)Cl]Cl和Ru(bpy)2Cl2所造成的影響之差異6........................................................60 3-3吡啶釕金屬錯合物與大腸桿菌作用所影響之蛋白質功能分 類..................................................62 第四章 結論..............................................63 參考文獻.......................................................65 圖表目錄 圖1.鉑金屬錯合物及其與鳥糞嘌呤 (guanine)之鍵結模型圖.......8 圖2.模擬蛋白質激酶抑制劑staurosporine之釕金屬錯合物結構圖........10 圖3.釕金屬錯合物與蛋白質鍵結之預測圖......................10 圖4.化學探針之結構圖......................................18 圖5.四種化學探針之作用機制................................19 圖6.化學探針在發展藥物上之實際應用........................19 圖7.蛋白質隨著環境pH值變化而改變淨電荷之曲線圖............24 圖8.等電焦集電泳之原理....................................24 圖9.Immobiline的結構式....................................25 圖10.ployacrylamide與immobiline共聚合之圖示...............25 圖11.DTT使蛋白質變性之圖示................................27 圖12.二維凝膠電泳之原理...................................27 圖13.IPG strip澎潤作用之圖示..............................41 圖14.一維凝膠電泳IEF之圖示................................42 圖15.IPG strip平衡作用之圖示..............................43 圖16.二維凝膠電泳SDS-PAGE之圖示...........................44 圖17.吡啶釕金屬錯合物之結構圖.............................70 圖18.吡啶釕金屬錯合物[Ru(terpy)(bpy)Cl]Cl對E. coli BL21(DE3)抑制之生長曲線............................................71 圖19. 加入[Ru(terpy)(bpy)Cl]Cl之2-D GEL...................72 圖20.加入Ru(bpy)2Cl2之2-D GEL.......................................................73 圖21.受吡啶釕金屬錯合物影響的蛋白質點分佈圖...............74 圖22.[Ru(terpy)(bpy)Cl]Cl up-regulated spots.........75 圖23.[Ru(terpy)(bpy)Cl]Cl up-regulated spot之蛋白質功能分佈圖...............................................75 圖24.[Ru(terpy)(bpy)Cl]Cl down-regulated spots........76 圖25.[Ru(terpy)(bpy)Cl]Cl down-regulated spots之蛋白質功能分佈圖...............................................76 圖26.[Ru(terpy)(bpy)Cl]Cl un-regulated spots.........77 圖27.[Ru(terpy)(bpy)Cl]Cl un-regulated spots之蛋白質功能分佈圖................................................77 圖28.Ru(bpy)2Cl2 up-regulated spots...................78 圖29.Ru(bpy)2Cl2 up-regulated spots之蛋白質功能分佈圖....78 圖30.Ru(bpy)2Cl2 down-regulated spots...................79 圖31.Ru(bpy)2Cl2 down-regulated spots之蛋白質功能分佈圖.......79 圖32.Ru(bpy)2Cl2 un-regulated spots.........80 圖33.Ru(bpy)2Cl2 un-regulated spots之蛋白質功能分佈圖........80 圖34.Distribution of protein function.................157 表1.[Ru(terpy)(bpy)Cl]Cl up-regulated spots之變化趨勢...........................81 表2.[Ru(terpy)(bpy)Cl]Cl up-regulated spots之生理功能...............................83 表3.[Ru(terpy)(bpy)Cl]Cl down-regulated spots之變化趨勢..........84 表4.[Ru(terpy)(bpy)Cl]Cl down-regulated spots之生理功能..................92 表5.[Ru(terpy)(bpy)Cl]Cl un-regulated spots之變化趨勢..........................103 表6.[Ru(terpy)(bpy)Cl]Cl un-regulated spots之生理功能..........................108 表7.Ru(bpy)2Cl2 up-regulated spots之變化趨勢.............................117 表8.Ru(bpy)2Cl2 up-regulated spots之生理功能.............119 表9.Ru(bpy)2Cl2 down-regulated spots之變化趨勢............................124 表10.Ru(bpy)2Cl2 down-regulated spots之生理功能..............131 表11.Ru(bpy)2Cl2 un-regulated spots之變化趨勢...............................141 表12.Ru(bpy)2Cl2 un-regulated spots之生理功能..............................148[[note]]學號: 693290222, 學年度: 9