臺灣博碩士論文加值系統

在台灣，因末期腎病（end-stage renal disease, ESRD） 須要接受長期透析或移植治療的患者，約有四萬多人。腎臟病在國人十大死因中佔第七位；而慢性尿毒症的排行中，每年新發生率方面，台灣皆佔世界的第二位。Continuous ambulatory peritoneal dialysis (CAPD) 是末期腎臟衰竭病患治療方式中較方便且不影響病患生活方式，但是長期腹膜透析所引起的腹膜纖維化（peritoneal fibrosis，PF）是腹膜透析病人最常見的合併症之一，嚴重時甚至會影響生命。臨床上發現：目前使用的傳統腹膜透析溶液 (peritoneal dialysis ，PD solution) 具有高糖、高滲透壓、低酸鹼度，等透析液生物相容性不佳的問題。其中高濃度糖為腹膜透析溶液中可成功調整滲透壓達到脫水目的最主要成分。然而，目前台灣常用的糖濃度為1.5％ (83.8mM)、2.5％ (138mM)及4.25％ (236mM)三種配製。人類腹膜間皮細胞 （human peritoneal mesothelial cell，HPMC） 長期暴露在這遠超過生理濃度的 glucose下會改變腹膜的結構與功能，造成人類腹膜間皮細胞逐漸脫落、細胞外間質(extracellular matrix ，ECM) 堆積、腹膜慢性發炎、間皮細胞的凋亡 （apoptosis），使腹膜剝離 （detachment）甚至功能衰敗。由先前的文獻指出 2.5%、4.25% glucose會引起人類腹膜間皮細胞壞死 (necrosis) 或細胞凋亡(apoptosis)，但是其分子機制引發HPMC目前仍未明瞭，尤其粒線體以及活性氧物質所扮演的角色及調控機制也尚未被釐清。本論文研究主旨為高糖引起人類腹膜細胞凋亡路徑中粒線體所扮演的角色，我們推測高糖處理會經由粒線體氧化磷酸化反應（oxidative phosphorylation）產生活性氧自由基 （reactive oxygen species，ROS） 而引發細胞凋亡，我們於本研究發現經過 138mM glucose 及 236mM glucose 處理的人類腹膜細胞會引起細胞凋亡;以西方墨點法可觀察到人類腹膜細胞 cytochrome c release 增加、 PARP cleavage增加以及 caspase–9 、 caspase-3被活化，並且增加 collagen mRNA 3.66倍的表現。此外，我們亦進一步觀察，以大鼠環間膜細胞（rat glomerular mesangial cell，RMC）為細胞模式的實驗中將 35mM glucose添加1mM l-N-acetylcystein (L-NAC)、粒線體氧化磷酸化抑制劑1μM rotenone ；及1μM粒線體去偶合劑carbonyl cyanide m-chlorophenylhydrazone (CCCP) 可降低高糖刺激對RMC造成的傷害及分別減低細胞凋亡50％、60％、12％。我們的實驗結果可以解釋糖尿病腎臟病的可能原因，以及高糖透析液對 CAPD 患者長期使用對腹膜的不良影響，也可以作為日後研發藥物治療的理論基礎。

In Taiwan, there are at present more than forty thousand patients with end-stage renal disease (ESRD) under regular dialysis therapy. Kidney disease is the seventh of ten leading causes of mortality in our country.The incidence rate of ESRD in Taiwan has gained the second place in the world. Continuous ambulatory peritoneal dialysis (CAPD) is a convenient therqpy as one kind of renal replacement therapy which interferes less of daily activity in ESRD patients. Peritoneal fibrosis (PF) is one of the most common complications in peritoneal dialysis (PD). It has been found that bio-incompatible PD solutions bearing characteristics of high glucose (HG), hyperosmolality, and low pH value may leads to the development of PF. The glucose concentration of PD solutions in Taiwan is 1.5％(83.8mM), 2.5％(138mM), or 4.25％(236mM). After long-term exposure of HG condition, human peritoneal mesothelial cells (HPMCs) will result in changes of peritoneal structure and function. Histological evaluations had demonstrated that HPMCs apoptosis and accumulation of extracellular matrix (ECM) are main pathogenesis of PF. However, the mechanisms of HG-induced apoptosis of HPMCs and the role of mitochondria as well as reactive oxygen species (ROS) in PF remains undetermined.
This research is aims to evaluate effects of HG in apoptosis of HPMCs. We also investigated the efficacy of mitochondria oxidative phosphorylation and ROS generation in HPMCs under HG condition. It was found that HG induced HPMC apoptosis, cytochrome c release, PARP cleavage, activations of caspase-9, caspase-3, and an up-regulated gene expression of type I collagen with 138mM and 236mM glucose treatment. It was also demonstrated that these detrimental effects of HG in RMC caould be significantly debased apoptosis 50％ by 1mM l-N-acetylcystein (L-NAC), debased apoptosis 60％ by 1μM rotenone or debased apoptosis 12％by 1μM (CCCP) carbonyl cyanide m-chlorophenylhydrazone supplemented. Induced apoptosis and ECM accumulation were also revealed in rat mesangial cell cuture with 35mM glucose treatment. Our work may have great clinical implications in management of diabetic nephropathy and provide pharmacological information for prevention of PF in long-term PD patients.

目錄
圖目錄 -----------------------------------------------------------------------------------------7
中文摘要 -------------------------------------------------------------------------------------10
英文摘要 -------------------------------------------------------------------------------------13
縮寫表 ----------------------------------------------------------------------------------------16
第一章 緒論：
壹﹑高糖引起細胞傷害--------------------------------------------------------------------20
貳﹑腎臟衰竭之介紹與治療：-----------------------------------------------------------20
一、腎臟衰竭分作--------------------------------------------------------------------------20
A.原發性腎絲球腎臟炎
B.急性腎絲球腎炎
C.急性腎衰竭
D.慢性腎衰竭
E.尿毒症
二、末期腎衰竭的治療：血液透析、腹膜透析及腎臟移植----------------------22
A. 血液透析
a.血液透析的優點
b.血液透析的缺點
c.有關血液透析的問題
B.腹膜透析(PD)
a.腹膜透析治療在臨床上的種類
b.適當透析
c.PD臨床上適用範圍及不適用範圍
d.PD注意事項
e.腹膜透析的優點
f.腹膜透析的缺點
g.有關腹膜透析的問題
C.腎臟移植
參、糖尿病腎病變的介紹
一、糖尿病腎病變的自然病程可分5期-----------------------------------------31
A.腎絲球高濾過率期
B.微量白蛋白尿期(microalbuminuria)
C.蛋白尿期(proteinuria)
D.腎衰竭期(end-stage renal disease)和尿毒期(uremia)
二、腎臟衰竭糖尿病患的處理
三、尿毒期糖尿病患的治療
四、糖尿病腎病變患者當已進入尿毒症時注意事項
肆、細胞凋亡--------------------------------------------------------------------------------34
一、細胞凋亡的特徵-----------------------------------------------------------------35
二、影響細胞凋亡的基因和凋亡蛋白（caspase）家族-----------------------35
三、腹膜透析液引發 HPMC apoptosis and oncosis----------------------------36
伍、粒線體
一、粒線體結構-----------------------------------------------------------------------37
二、粒線體的功能--------------------------------------------------------------------37
三、Cytochrome c 由粒線體釋放引起細凋亡----------------------------------38
四、腹膜透析液引發HPMC粒線體DNA傷害-------------------------------38
陸、活性氧自由族(reactive oxygen species, ROS)
一、自由基(Free radical)的由來與特性------------------------------------------39
二、ROS的功能與影響-------------------------------------------------------------39
三、腹膜透析液引起HPMC的ROS 產生增加-------------------------------40
第二章 研究目的 ------------------------------------------------------------------------41
第三章 實驗的材料與方法---------------------------------------------------------------43
壹﹑實驗材料 ------------------------------------------------------------------------------44
貳﹑研究方法
一、人類腹膜間皮細胞培養與鑑定----------------------------------------------48
二、大鼠環間膜細胞的取得與初代培養----------------------------------------48
三、細胞存活率(Cell viability) 試驗--------------------------------------------49
四、細胞蛋白質萃取液製備 (Preparation of cell lysate) ---------------------51
五、蛋白質定量法 (Protein assay) -----------------------------------------------51
六、西方墨點法(western blotting) ------------------------------------------------52
七、流式細胞儀分析----------------------------------------------------------------53
八、粒線體產生ATP能力測定---------------------------------------------------54
九、脂質過氧化物(lipoperoxide)的測定：MDA法---------------------------54
十、北方墨點法(Northern blotting) ----------------------------------------------56
十一、TUNEL and DAPI stain-----------------------------------------------------58

第四章 研究結果
壹﹑以MTT assay 和 dye exclusion 分析不同劑量的 glucose 對人類腹膜間皮細胞 (HPMC) 存活率的影響-------------------------------------------------------61
貳﹑利用北方點墨法 (Northern blot) 分析人類腹膜細胞 (HPMC) 、大鼠環間膜細胞 （RMC） 細胞內膠原蛋白基因的表現-----------------------------------61
参、Glucose引發人類腹膜間皮細胞 (HPMC)中活性氧化物(ROS)增加-------62
肆﹑Glucose造成人類腹膜間皮細胞(HPMC)脂質傷害-----------------------------62
伍、Glucose 影響 HPMC 與 RMC 細胞粒線體的ATP形成能力--------------63
陸、在細胞培養實驗模式觀察正常人類腹膜間皮細胞 (HPMC)、大鼠環間膜細胞（RMC）在不同濃度的糖溶液下引發細胞凋亡的調控 -------------------63
柒、以西方點墨法分析 HPMC 內 apoptosis 訊息傳遞路徑活性的變化------64
捌、經由大鼠環間膜細胞培養實驗觀察 ROS 抑制劑、氧化磷酸化的去偶合劑、 complex I 抑制劑與細胞生存力的相關性----------------------------------------65
玖、經由大鼠環間膜細胞培養實驗觀察 ROS 抑制劑、氧化磷酸化的去偶合劑、 complex I 抑制劑與活性氧化物 (ROS) 表現的相關性-----------------------66
拾、經由大鼠環間膜細胞培養實驗觀察ROS抑制劑、氧化磷酸化的去偶合劑、complex I 抑制劑與膠原蛋白基因表現的相關性-------------------------------67
拾壹、經由大鼠環間膜細胞培養實驗觀察 ROS 抑制劑、氧化磷酸化的去偶合劑、 complex I 抑制劑與細胞凋亡表現的相關性--------------------------67
第伍章 討論----------------------------------------------------------------------------------68
第陸章 未來展望----------------------------------------------------------------------------74
第七章 圖表----------------------------------------------------------------------------------77
第捌章 參考文獻---------------------------------------------------------------------------100

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