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研究生:葉怡倩
研究生(外文):Yi-Chien Yeh
論文名稱:連續腹膜透析的末期腎病病患罹患腹膜炎之腹膜炎引流物刺激心肌細胞引起細胞凋亡之探討
論文名稱(外文):Study the Apoptotic Effect of Dialysis Peritonitis Effluents from ESRD Patients in Cultured Cardiomyocytes
指導教授:黃昭菱黃昭菱引用關係
指導教授(外文):Jau-Ling Huang
學位類別:碩士
校院名稱:長榮大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:103
中文關鍵詞:末期腎病連續腹膜透析腹膜炎細胞凋亡
外文關鍵詞:ESRDCAPDPeritonitisApoptosisGATA-4Bcl-2Bax
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心臟疾病是長期連續腹膜透析的末期腎病患者主要致死的原因。反覆罹患腹膜炎的長期連續腹膜透析病患,心臟疾病死亡的比率更是高於一般長期連續腹膜透析的病患,導致病患存活率下降。本論文主要探討反覆感染腹膜炎之連續腹膜透析的病患其腹膜炎引流物與心肌病變的相關性。以培養之大鼠心肌細胞(H9c2)實驗模式,細胞存活分析實驗證實腹膜炎引流物會對心肌細胞產生毒性並造成死亡且具有濃度相關性。利用流式細胞儀分析腹膜炎引流物對於心肌細胞之影響,發現其細胞週期會停滯在G2 / M時期並且也觀察到sub-G1時期有增加的情況。同時利用磷脂結合蛋白(Annexin V)去分析腹膜炎引流物是否造成細胞凋亡,結果顯示腹膜炎引流物會透過細胞凋亡機制而造成心肌細胞死亡。以即時定量反轉錄酶聚合酶連鎖反應(Real-time RT-PCR)以及西方墨點法(Western blot)分析,結果顯示腹膜炎引流物會使與心臟存活有關的重要轉錄因子GATA-4表現量下降,因而影響GATA-4所調控的下游調控者Bcl-2,使得其表現量下降。此外透過西方墨點法分析,發現腹膜炎引流物會誘導細胞凋亡調控者Bax表現量增加。因此本論文推論腹膜炎引流物是一種具有心肌毒性的物質,會透過抑制GATA-4、Bcl-2表現及誘導Bax增加而干擾Bax / Bcl-2比值,使得心肌細胞走向細胞凋亡而造成心肌損傷,因而導致反覆感染腹膜炎的連續腹膜透析病患更容易因為心臟產生病變而死亡。
Cardiac disease is the leading cause of death among patients receiving long-term continuous ambulatory peritoneal dialysis(CAPD), especially in patients with frequent peritonitis. We proposed that the peritonitis toxins might enter the systematic circulation, and lead to heart diseases. The purpose of this study was to explore whether the peritonitis effluents of end-stage renal disease(ESRD)patients theraping with CAPD could induce cytotoxicity in H9c2-cultured cardiomyoblast. The results showed a dose-dependent cell death in H9c2 cells treated with peritonitis effluents. The effect of peritonitis effluents in H9c2 cells was studied by flow cytometric analysis. The results of proidium iodide stain showed that the percentage of the cells arrested at G2/M phase were obviously higher then that of untreated cells. The proidium iodide stain also showed that the peritonitis effluent could increase the cells of sub-G1 phase. Annexin V analysis furthere demonstrated that peritonitis effluents could induce an apoptosis response. Real-time RT-PCR and western blot analysis showed that the peritonitis effluents could reduce the expression of GATA-4, a cardiac important transcription factor, and then to affect the expression level of its downstream target Bcl-2, the anti-apoptotic regulator. The western blot also showed that the peritonitis effluents could increase the expression of pro-apoptosis regulator Bax in H9c2 cells. These results suggested that peritonitis effluents could be cardiotoxic and function as an apoptotic inducer. Peritonitis toxins might through perturb the ratio of Bax / Bcl-2 through GATA-4 transcription factor to cause the death of cardiomyocytes.
目 錄
中文摘要 1
英文摘要 3
重要英文名詞縮寫對照 5
第一章緒論 7
第一節腎臟病、透析之流行病學研究 7
第二節透析治療方法之比較 9
第三節腹膜透析病患主要致病與死亡因素-心肌損傷死亡、心臟衰竭 9
第四節反覆腹膜炎感染的腹膜透析病患與心肌損傷死亡之相關性 10
第五節引起心肌損傷死亡、心臟衰竭相關機制探討-心肌凋亡以及參與之訊息傳遞分子 11
第六節GATA-4轉錄因子與Bcl-2家族相關探討 13
第七節研究目的與方向 15
第二章研究材料與方法 17
第一節實驗試劑 17
第二節實驗儀器 19
第三節實驗抗體 20
第四節實驗溶液配製 20
第五節 實驗藥物準備與處理 25
第六節細胞株培養、繼代以及細胞計數與種細胞 27
第七節細胞保存與解凍 29
第八節細胞存活率分析 30
第九節心肌細胞之收集 31
第十節細胞週期之分析 32
第十一節細胞凋亡之分析 33
第十二節全量核醣核酸之抽取與濃度定量 35
第十三節反轉錄酶反應 36
第十四節引子設計 37
第十五節即時聚合酶鏈連鎖反應 37
第十六節蛋白質處理與濃度定量 38
第十七節蛋白質分析 39
第十八節分析與統計方法 41
第三章結果 42
第一節腹膜炎引流物對心肌細胞的影響 42
第二節心肌細胞受腹膜炎引流物刺激後其細胞週期的變化 44
第三節腹膜炎引流物主要透過細胞凋亡路徑造成心肌細胞死亡 46
第四節GATA-4轉錄因子可能參與腹膜炎引流物所引起的心肌細胞凋亡機制 47
第五節腹膜炎引流物可能透過GATA-4轉錄因子調控Bcl-2 family來誘導細胞凋亡活化引起心肌細胞死亡 49
第六節腹膜炎引流物細胞毒殺特異性 51
第四章討論 53
第一節腹膜炎引流物對心肌細胞的影響探討 53
第二節腹膜炎引流物對心肌細胞的細胞週期與細胞凋亡之影響 54
第三節GATA-4轉錄因子、Bcl-2 family與腹膜炎引流物探討 55
參考文獻 59
表目錄 68
表一、慢性腎臟疾病嚴重程度分類表 68
表二、慢性腎臟疾病嚴重程度與罹患心臟疾病風險機率表 69
表三、罹患腹膜炎的腹膜透析病患造成死亡的危險因子 70
表四、腹膜炎引流物檢體相關微生物培養之資料 71
表五、引子序列 72
圖目錄 73
圖一、透析病人與正常族群在心臟疾病死亡率上比較 73
圖二、透析病人有無伴隨心臟衰竭與存活率有明顯相關 74
圖三、罹患腹膜炎的次數多寡會影響透析病人的存活率 75
圖四、實驗流程 76
圖五、光學顯微鏡下觀察大鼠心肌母細胞之細胞形態 77
圖六、心肌細胞處理藥物後細胞之形態改變及死亡 78
圖七、腹膜炎引流物濃度梯度對心肌細胞存活率的影響(一)79
圖八、腹膜炎引流物濃度梯度對心肌細胞存活率的影響(二)80
圖九、腹膜炎引流物作用不同時間對心肌細胞存活率的影響 81
圖十、腹膜炎引流物誘導細胞週期停滯於G2/M期(一)82
圖十一、腹膜炎引流物誘導細胞週期停滯於G2/M期(二)83
圖十二、腹膜炎引流物誘導心肌細胞sub-G1期增加(一)84
圖十三、腹膜炎引流物誘導心肌細胞sub-G1期增加(二)85
圖十四、腹膜炎引流物會刺激心肌細胞引發細胞凋亡(一)86
圖十五、腹膜炎引流物會刺激心肌細胞引發細胞凋亡(二)87
圖十六、即時定量PCR相對定量GATA-4訊息核醣核酸之擴增情形圖
(Amplification plot)88
圖十七、腹膜炎引流物誘導心肌細胞GATA-4訊息核醣核酸表現量下降 89
圖十八、腹膜炎引流物處理不同時間,GATA-4訊息核醣核酸表現量之情形 90
圖十九、腹膜炎引流物誘導心肌細胞GATA-4蛋白質表現下降 91
圖二十、腹膜炎引流物處理不同時間後GATA-4蛋白質表現量之量化結果 92
圖二十一、即時定量PCR相對定量Bcl-2訊息核醣核酸之擴增情形圖
(Amplification plot)93
圖二十二、腹膜炎引流物誘導心肌細胞內Bcl-2訊息核醣核酸表現量下降 94
圖二十三、腹膜炎引流物處理不同時間,Bcl-2訊息核醣核酸表現量之情形 95
圖二十四、腹膜炎引流物誘導心肌細胞Bcl-2蛋白質表現下降 96
圖二十五、腹膜炎引流物不影響心肌細胞Bcl-XL蛋白質表現 97
圖二十六、腹膜炎引流物誘導心肌細胞Bax蛋白質表現增加 98
圖二十七、腹膜炎引流物誘導心肌細胞Bax / Bcl-2蛋白質比值增加 99
圖二十八、光學顯微鏡下觀察骨肉瘤細胞株細胞形態 100
圖二十九、光學顯微鏡下觀察骨肉瘤細胞處理過藥物後細胞形態 101
圖三十、MTT法分析腹膜炎引流物對骨肉瘤細胞存活影響 102
附錄 103
附錄一、第二十二屆生物醫學聯合學術年會口頭論文報告摘要 103
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