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研究生:吳曉怡
研究生(外文):Hsiao-Yi Wu
論文名稱:糖尿病高血糖導致大鼠腎臟腎絲球肥大的機制探討
論文名稱(外文):The mechanisms of diabetic hyperglycemia-induced rat renal glomerular hypertrophy
指導教授:劉興華劉興華引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:毒理學研究所
學門:醫藥衛生學門
學類:其他醫藥衛生學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:70
中文關鍵詞:高血糖腎絲球肥大
外文關鍵詞:hyperglycemiaglomerularhypertrophy
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在美國,糖尿病腎病變不但是糖尿病微血管的併發症之一,而且最後會導致末期腎病變。在第一型糖尿病的病人會有20%到40%以及第二型糖尿病病人會有10到20%會發展成腎病變。糖尿病腎病變早期的病理變化會造成腎絲球肥大、腎絲球環間膜細胞擴張、腎絲球基底膜變厚以及細胞外基質蓄積,隨著病程的發展最後會導致腎絲球硬化,甚至造成蛋白尿及腎衰竭。在動物及細胞的實驗模式,高血糖和高濃度葡萄糖狀態下,會導致腎絲球細胞肥大及細胞外基質蓄積。但高血糖造成腎絲球肥大的分子機制仍不清楚。因此本實驗研究目的在探討高血糖狀態下,造成腎絲球肥大的訊息傳遞路徑為何。
我們首先的實驗是探討在細胞模式下,高濃度葡萄糖對腎絲球肥大的影響。在分離的腎絲球外加高濃度葡萄糖,會增加蛋白質/DNA的比值、腎絲球體積、自由基產生、PGE2含量以及fibronectin表現增加。同時也觀察到高濃度葡萄糖會促使腎絲球Akt磷酸化、活化NF-κB、COX-2、TGF-β1 以及p27kip1 表現增加。NAC(抗氧化劑)、LY294002 (PI3K 抑制劑)和NS398 (COX-2 抑制劑)可以有效抑制蛋白質/DAN 比值和腎絲球體積增加。
另外,在糖尿病動物模式下腎絲球肥大的影響。我們實驗證明糖尿病兩週,腎臟體重比值、腎絲球蛋白質DNA 比值、腎絲球體積以及血清中BUN 和Creatinine 增加。同時也觀察到糖尿病大鼠會增加腎絲球Akt 磷酸化、COX-2、fibronectin 以及p27kip1 表現。NAC(抗氧化劑)、和Meloxicam(COX-2 抑制劑)可以有效抑制腎臟和體重比值、蛋白質和DAN 比值以及腎絲球體積增加。
綜合以上結果,腎臟的腎絲球在高血糖狀態下,導致腎絲球肥大的訊息傳遞路徑是活化PI3K 使ROS 含量增加,而活化Akt,藉由NF-κB-COX-2-PGE2 的表現,促使TGF-β1 表現增加,而誘發cyclin-dependent kinase (CDK) inhibitor p27kip1 表現。這一訊息傳遞路徑在高血糖導致腎絲球肥大中佔有重要的角色。
Diabetic nephropathy (DN) is one of the most devastating microvascular complications of diabetes as well as the leading cause of end-stage renal disease (ESRD) in the United States. Approximately 20 to 40% of the patients with type 1 diabetes and 10 to 20% of those with type 2 diabetes develop nephropathy. The earliest pathologic changes of DN are characterized by glomerular hypertrophy, mesangial expansion with the thickening of glomerular basement, expanded extracellular matrix (ECM), and glomerular sclerosis, which ultimately cause the progression of proteinuria and renal failure. Data from animal models as well as cultured renal cells the hyperglycemia and high glucose induced hypertrophy and extracellular matrix expansion. The molecular mechanisms of hyperglycemia-induced glomerular hypertrophy remain unknown.
In the present study, we first examined the effects of high glucose on glomerular hypertrophy in vitro. We found that isolated renal glomeruli treated with high glucose enhanced the protein/DNA ratio, glomerular volume, reactive oxygen species (ROS) production, PGE2 production and fibronectin expression. Moreover, high glucose also triggered the phosphorylation of Akt, activation of nuclear factor-κB, cyclooxygenase-2 (COX-2) expression, transforming growth factor-β1 (TGF-β1) expression, and p27kip1 expression in isolated renal glomeruli. Antioxidant N-acetylcysteine, phosphoinositide 3-kinase inhibitor LY294002, and COX-2 inhibitor NS398 significantly reversed the high glucose-induced increase of protein/DNA ratio, and glomerular volume.
On the other hand, we examined the effect of hyperglycemia on glomerular hypertrophy in vivo. We found that enhanced the kidney weight/body weight ratio, protein/DNA ratio, glomerular volume, BUN, and creatinine were increased in isolated glomeruli of streptozotocin -diabetic rats. Moreover, the phosphorylation of Akt, cyclooxygenase-2
(COX-2) expression, fibronectin expression, and p27kip1 expression in isolated glomeruli of diabetic rats were also triggered. Treatment with antioxidant N-acetylcysteine and COX-2 inhibitor meloxicam significantly reversed the increased in kidney weight/body weight ratio, protein/DNA
ratio, and glomerular volume in isolated glomruli of diabetic tars.
In conclusion, these results indicate that ROS/Akt/NF-κB/COX-2/PGE2/TGF-β1/ p27kip1signaling pathway may play an important role in hyperglycemia-induced renal glomeruli hypertrophy.
中文摘要 ………………………………………………………………1
英文摘要 ………………………………………………………………3
第一章
第一節 糖尿病 ………………………………………………………5
第二節 糖尿病流行病學 ……………………………………………6
第三節 糖尿病併發症-腎病變 .............................7
第四節 糖尿病腎絲球肥大可能的分子機制 ................ 9
第二章
第一節 動物處理 ………………………………………………… 13
第二節 基礎生理生化值測量 …………………………………… 15
第三節 腎絲球的分離 …………………………………………… 15
第四節 細胞核蛋白的萃取................................16
第五節 蛋白質分析西方墨點法............................17
第六節 mRNA分析 ……………………………………………… 20
第七節 腎絲球ROS含量測定 …………………………………… 22
第八節 蛋白質和DNA的比值..............................23
第九節 腎絲球體積的量化 ……………………………………… 23
第十節 實驗數據之統計……………………………………………24
第三章
第一節 PartⅠ 高糖狀態下對大鼠腎絲球分離後導致腎絲球肥大的影響 ………………………………………………………29
第二節 PartⅡ 糖尿病大鼠導致腎絲球肥大的影響 ………35
第四章
第一節 糖尿病初期大鼠腎臟組織病變的發展............39
第二節 ROS在糖尿病初期導致腎絲球肥大的角色.........40
第三節 Akt的活化在糖尿病初期導致腎絲球肥大的角色...41
第四節 NF-kB、COX-2和PGE2在糖尿病初期腎絲球肥大角色42
第五節 Fibronectin、TGF-B1和p27 kip1在糖尿病初期導致腎絲球肥大的角色..........................................43
第六節 結論..........................................44
參考文獻 ………………………………………………………………45
圖表 ……………………………………………………………………53
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