前言:糖尿病腎病變(diabetic nephropathy)是造成已開發國家末期腎臟疾病(end-stage renal disease)的主要原因，在眾多糖尿病腎病變腎絲球的型態表現中，足細胞(podocyte)數目減少為預測糖尿病腎病變進展的重要因子，而足細胞凋亡會引起足細胞掉落。細胞質水解酵素A2 (cPLA2α) 會引起細胞凋亡、感染、發炎等現象。本實驗想要探討cPLA2α是否有參與高糖刺激足細胞凋亡的過程及其機制。
方法和材料:大鼠足細胞培養於正常糖(glucose 5.5mM)或高糖(glucose 25mM)的環境，利用免疫螢光染色、流式細胞儀、西方墨點法來觀察活性氧化物(reactive oxygen species, ROS)、凋亡現象和caspase蛋白質表現；另外在高糖組加入cPLA2α抑制劑(AACOCF3) (10μM)，觀察AACOCF3在ROS、凋亡現象和caspase蛋白質表現的角色。
結果: 高糖刺激1小時後，phospho-cPLA2α/cPLA2α ratio開始增加，(control to 1 hour : 0.75±0.14 to 1.69±0.08，P<0.01)，phospho-cPLA2α /cPLA2α ratio隨著時間增加 (control to 24 hour : 0.75±0.14 to 2.57±0.31，P<0.01)；經過高糖刺激1小時，足細胞內ROS開始產生，出現凋亡，高糖持續刺激24小時後，大量ROS產生(control to 24 hour : 0.66±0.33 to 7.48±2.23，P<0.05)，凋亡足細胞更是增加(control to 24 hour : 0.75±0.2 to 4.83±0.9，P<0.05)，而caspase 3和caspase 9明顯活化(cleaved caspase 3 : control to 24 hour : 0.75±0.15 to 3.83±0.50，P<0.001；cleaved caspase 9 : control to 24 hour : 0.65±0.15 to 3.39±0.46，P<0.001)，不過caspase 8對高糖的刺激沒有明顯活化反應。接著觀察cPLA2α inhibitor (AACOCF3)的影響力，在經過高糖刺激24小時後，給予AACOCF3會抑制足細胞ROS產生(HG to HG+ AACOCF3 : 3.21±0.49 to 1.34±0.08，P<0.05)和凋亡(HG to HG+ AACOCF3 : 1.76±0.20 to 1.13±0.06，P<0.05)；減少caspase 3 (HG to HG+ AACOCF3 : 2.41±0.32 to 0.96±0.12，P<0.05)和9 (HG to HG + AACOCF3 : 4.76±0.35 to 2.1±0.19，P<0.01)的活化；抑制了cytochrome c的轉移到細胞質(HG to HG + AACOCF3 : 8.13±0.66 to 1.82±0.24，P<0.001)，減少因高糖刺激而增加的 Bax (HG to HG+ AACOCF3 : 2.26±0.28 to 0.67±0.12，P<0.01)，增加了因高糖刺激而減少的Bcl-2(HG to HG+ AACOCF3 : 0.77±0.1 to 1.34±0.06，P<0.01)。
結論: 由本實驗可證實高糖刺激足細胞凋亡的路徑為intrinsic mitochondrial proapoptotic pathway，cPLA2α經由mitochondria -dependent cascade參與高糖刺激足細胞凋亡機制。

Introduction: Podocyte depletion and apoptosis was the earliest phenomenon of progression of diabetic nephropathy. cPLA2α wound induce apoptosis in various cell model. The aim of this study was to investigate the pathway of podocyte apoptosis induced by high glucose and the role of cPLA2α in high glucose-stimulated podocyte apoptosis.
Methods & Materials: Primary rat podocytes were treated with high glucose (25mM) and AACOCF3 (10μM). Immunofluorescent studies, flow cytometry and western blotting were utilized for analysis cPLA2α phosphorylation, ROS production, apoptosis and caspase protein.
Results: After the stimulation of high glucose, cPLA2α phosphorylatio was increased at 1 hour (control to 1 hour : 0.75±0.14 to 1.69±0.08，P<0.01) and increased progressively up to 24 hours (control to 24 hour : 0.75±0.14 to 2.57±0.31，P<0.01). ROS production and apoptosis were observed at 1 hour and were augmented progressively up to 24 hours (ROS : control to 24 hour : 0.66±0.33 to 7.48±2.23，P<0.05；apoptosis : control to 24 hour : 0.75±0.2 to 4.83±0.9，P<0.05). High glucose induced the increase level of caspase 3 (control to 24 hour : 0.75±0.15 to 3.83±0.50，P<0.001) and 9 (control to 24 hour : 0.65±0.15 to 3.39±0.46，P<0.001), not caspase 8 at 24 hours. AACOCF3 attenuated ROS (HG to HG+ AACOCF3 : 3.21±0.49 to 1.34±0.08，P<0.05) production and apoptosis (HG to HG+ AACOCF3 : 1.76±0.20 to 1.13±0.06，P<0.05) induced by high glucose. The increase of caspase 3 (HG to HG+ AACOCF3 : 2.41±0.32 to 0.96±0.12，P<0.05), 9 (HG to HG + AACOCF3 : 4.76±0.35 to 2.1±0.19，P<0.01), cytosol cytochrome c (HG to AACOCF3 : 8.13±0.66 to 1.82±0.24, P<0.001) and Bax (HG to HG+ AACOCF3 : 2.26±0.28 to 0.67±0.12，P<0.01) induced by high glucose was mitigated after treatment of AACOCF3. AACOCF3 increased the diminished level of Bcl-2 elicited by high glucose (HG to HG+ AACOCF3 : 0.77±0.1 to 1.34±0.06，P<0.01).
Conclusion: High glucose-stimulated apoptosis in podocytes is mediated through intrinsic mitochondrial proapoptotic pathway. cPLA2α contributes to podocyte apoptosis induced by high glucose via mitochondria-dependent cascade.

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英文摘要------------------------------------------- p.6
前言 ------------------------------------------------ p.8
研究材料與方法---------------------------------- p.12
研究結果------------------------------------------- p.19
討論 ------------------------------------------------ p.24
結論 ------------------------------------------------ p.29
圖表 ------------------------------------------------ p.30
參考文獻------------------------------------------- p.45

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