臺灣博碩士論文加值系統

中文摘要............................................................................................................I
英文摘要.........................................................................................................III
目錄..................................................................................................................IV
第一章 緒論 (Introduction)…..………………………………….……….1
背景………………………………………………………….…..............2
第二章 材料與實驗方法………………………………….………………..9
第一節 材料…………………………………………….……………...10
一、藥品試劑 ………………………………………………………10
二、常用儀器 ……………………………………………….………15
三、常用溶液 ………………………………….…….……..………17
第二節 實驗方法...…………………………….………………………19
一、細胞培養(cell culture) ……………………………….……….20
二、RNA萃取...................................................................................21
三、反轉錄 (Reverse Transcription) ..............................................21
四、聚合酶連鎖反應 (Polymerase chain reaction，PCR)...........21
五、即時聚合酶鏈鎖反應(Real-time PCR) ....................................22
六、免疫組織染色 (Immuo-histochemistry Staining，IHC) ......23
七、免疫螢光染色(Immuno-flurorescent Stain，IF) ..................24
八、細胞蛋白溶解物(cell lysate)的萃取............................................25
九、蛋白質定量..................................................................................25
十、蛋白質電泳凝膠(SDS-PAGE)的配製.........................................26
十一、SDS-PAGE蛋白質膠體電泳...................................................26
十二、西方墨點法 (Western blotting assay) ...................................27
十三、尼古丁低濃度血清培養..........................................................28
十四、small interfering RNA (si-RNA)技術....................................28
十五、CMV promoter基因放大技術................................................29
十六、Promoter Luciferase Assay...................................................30
十七、接合反應(ligation)及菌種轉型(Transformation) ...............31
十八、選養轉型成功的菌落並製備質體.........................................31
十九、菌種保存、限制酶切割確認及定序......................................32
二十、抽大量質體及轉染 (Transfection) ......................................32
二十一、細胞存活率測定 (MTT cell viability assay) ...................33
二十二、免疫沉澱法(Immuno-precipitation, IP) ..........................33
二十三、以組織研磨器分離細胞核、細胞質及粒線體..................35
二十四、統計分析方法......................................................................37
第三章 實驗結果(Result)...……………………………………….………38
第一節 人類乳癌細胞株及正常乳房上皮細胞株中PPM1F蛋白表現型態.....................................................................................................39
第二節 人類乳癌細胞株的PPM1F蛋白表現型態，以及乳癌細胞株中荷爾蒙受體與生長因子接受器的表現情形................................39
第三節 台灣乳癌病人腫瘤組織及正常乳房組織中PPM1F基因mRNA表現型態................................................................................40
第四節 以Real-time PCR比較台灣乳癌病患其腫瘤組織及正常乳房組織中PPM1F基因mRNA的表現程度........................................41
第五節 以統計軟體分析台灣乳癌病患其腫瘤組織及正常乳房組織中PPM1F基因mRNA的表現程度與臨床病理因子(clinical pathology factor)之間的關係...........................................................42
第六節 台灣乳癌病患腫瘤組織中PPM1F基因mRNA表現程度不論在絕對表現量或相對倍數皆高於正常乳房組織中的表現........................................................................................................43
第七節 台灣乳癌病患檢體中，腫瘤大小越大其PPM1F基因mRNA表現量越高........................................................................................44
第八節 台灣乳癌病患癌症期別越後期者其PPM1F的mRNA表現量越高…………………………………………………………………45
第九節 PPM1F mRNA表現量越高對於乳癌病患術預後存活越不利........................................................................................................45
第十節 暴露於尼古丁(Nicotine)環境中會促使PPM1F 基因mRNA表現量上升........................................................................................46
第十一節 在乳癌組織切片中PPM1F蛋白的表現形態...................47
第十二節 利用Si-RNA (small interfering RNA)抑制人類乳癌細胞株中的PPM1F.....................................................................................48
第十三節 抑制乳癌細胞MDA-MB-231中PPM1F的表現量並觀察其生長速率...........................................................................................48
第十四節 在低血清濃度環境培養下加入尼古丁(Nicotine)會促使PPM1F 基因mRNA及蛋白表現量上升......................................49
第十五節 尼古丁(Nicotine)的刺激會促進PPM1F基因啟動子(promoter)的活化...........................................................................51
第十六節 PPM1F蛋白在人類乳癌細胞株及正常乳房上皮細胞株中分布情形不同.................................................................................53
第十七節 分離乳癌細胞的細胞核、粒線體以及細胞質並觀察PPM1F表現情形.........................................................................................54
第十八節 乳癌細胞株中PPM1F蛋白表現的分布情形會因受到尼古丁刺激而改變.................................................................................55
第二十節 將暴露於尼古丁環境的乳癌細胞細胞核、粒線體以及細胞質分離並觀察PPM1F蛋白表現情形.............................................56
第二十一節 使乳癌細胞株中PPM1F蛋白過度表現並觀察乳癌細胞生長速率............................................................................................56
第四章 討論(Discussion)..............................................................................61
第五章 圖表(Tables and Figures)...............................................................68
第六章、參考資料(Reference)....................................................................97
第七章、附錄(appendix)............................................................................102

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