Hyperosmolarity enhanced the fibrosis susceptibility by phosphorylation of type II TGF-β receptors and up-regulation of Tbeta-RI in MDCK cells

中文摘要 i 英文摘要 iii 致謝 v 縮寫表 vii 第一章 緒論 一、糖尿病與糖尿病腎病變之盛行率 1 二、甘露醇（D -mannitol）簡介 2 三、腎纖維化之致病機轉 3 四、乙型轉型因子以及乙型轉型因子受器結構與命名 4 五、乙型轉型生長因子之作用與調節 6 六、腎纖維化與乙型轉型生長因子受器之作用 7 七、纖維化與乙型轉型生長因子之訊息傳導路徑 8 八、蛋白質降解之泛素蛋白酶體降解途徑 12 九、研究目的 14 第二章 材料與方法 一、試劑與材料 15 二、方法 16 1. 細胞培養 16 (1) 繼代細胞 16 (2) 活化細胞 17 (3) 凍細胞 17 2. 細胞數目 17 3. 細胞存活率 18 4. 酵素連結免疫吸附分析(ELISA) 18 5. 總量核酸核酸萃取及cDNA的製備 18 6. 反轉錄聚合酵素連鎖反應(RT-PCR) 19 7. 瓊脂膠體電泳 20 8. 細胞內第二型乙型轉型生長因子受器磷酸化分析 20 9. 細胞蛋白質總量測定 21 10.西方墨點分析 21 11.免疫沉澱法 23 12.統計分析 24 第三章 結果 一、高滲透壓以及乙型轉型生長因子對纖維蛋白之影響 25 二、高滲透壓對乙型轉型生長因子之影響 26 三、高滲透壓對乙型轉型生長因子接受器之影響 26 1、高滲透壓對乙型轉型生長因子受器蛋白質表現之影響 26 2、高滲透壓對第二型乙型轉型生長因子受器磷酸化之影響 27 四、高滲透壓增加第一型乙型轉型生長因子受器蛋白質之探討 27 五、高滲透壓對乙型轉型生長因子受器蛋白質上Smad 7- Smurf l 之影響 28 六、高滲透壓導致第一型乙型轉型生長因子受器蛋白質半衰期增加 29 七、高滲透壓對乙型轉型生長因子訊息傳導路徑-Smad途徑之影響 30 第四章 討論 31-36 第五章 圖表 36-45 第六章 參考文獻 46-53 參考文獻 Ateshian, G. A., Soltz, M. A., Mauck, R. L., Basalo, I. M., Hung, C. T. & Lai, W. 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Kidney Int 54(suppl): S10-13, 1996. 楊景民、郝麗瑾 (1995) 滲透壓及其臨床應用。荷澤醫專學報，7 (1)：3-6。[[abstract]]根據衛生署2006年所公佈的資料顯示，糖尿病為95年國人十大死因的第四名。高血糖高滲透壓狀態 (簡稱HHS) 是糖尿病最嚴重的急性併發症之一，其死亡率居高不下。 乙型轉型生長因子(TGF-β)是一個纖維激素，與腎臟纖維化的進展有相關，而隨後活化乙型轉型生長因子受器(TGF-βRII和TGF-βRI)，造成細胞外基質蛋白堆積，導致腎臟的纖維化。在這研究當中，我們利用甘露醇誘發高滲透壓來證明在遠端腎小管細胞中第二型乙型轉型生長因子受器磷酸化和第一型乙型轉型生長因子受器表現。我們使用狗腎遠端小管(MDCK)細胞，將細胞培養於不同滲透壓濃度的情況。 在高滲透壓下，經過乙型轉型生長因子(10ng/ml)之處理，藉由酵素連結免疫吸附分析來觀察纖維蛋白表現量。使用西方墨點法來研究乙型轉型生長因子受器以及下游蛋白質的表現。我們發現在甘露醇會提升乙型轉型生長因子所誘導纖維蛋白增加，然而，在滲透壓濃度劑量及時間點下，遠端小管細胞第二型乙型轉型生長因子受器上酪胺酸336位置磷酸化。高滲透壓下也會導致第一型乙型轉型生長因子受器以及乙型轉型生長因子下游訊息路徑Smad的表現。另外，使用cyclohexamide刺激，我們發現高滲透壓增加第一型乙型轉型生長因子受器半衰期，並且使抑制第一型乙型轉型生長因子受器走向 ubiquitination – proteosomal水解路徑。由以上這些結果來看，遠端腎小管細胞在高滲透壓會提升纖維化的敏感性，藉由第二型乙型轉型生長因子受器磷酸化以及第一型乙型轉型生長因子受器表現。 Diabetes mellitus have taken the fourth place in the ten major causes of the death of according to the reports issued by the Department of Health in Taiwan, 2006. Hyperglycemic hyperosmolar state (HHS) is one of the most serious acute complications of diabetes and its mortality rate had been higher. Transforming growth factor beta 1(TGF-β1) is a fibrokine implicated in the progression of renal fibrosis, followed TGF-β1 receptor (TGF-β RII and TGF-β RI) activation resulting in accumulation of extracllular matrix protein and caused renal cellular fibrosis. In this study, we utilized mannitol-inducing hyperosmolarity to elucidate the phosphorylation of type II TGF-β receptors and expression of Tbeta-RI in distal renal tubular cells. We were used Madin-Darby Canine Kidney (MDCK) cells, cultured in the presence of various on concentration of osmotic. Hyperosmolarity induce fibronectin protein level by TGF-β1(10ng/ml) using enzyme-linked immunosorbent assay. Western blotting were used to investigate the protein expression of TGF-βreceptors and downstream protein. We showed that mannitol enhanced TGF-β–induced increase in fibronectin in MDCK cells. Moreover, Osmotic concentration time- and dose- dependently induced the phosphorylation of Tbeta RII in tyrosine 336 in MDCK cells. Hyperosmolarity also induced increase expression of Tbeta RI and Smad signal pathway. In addition, by using cyclohexamide, we showed that hyperosmolarity significantly increased down-regulated the half-life and inhibited protein levels of TbetaRI by poly-ubiquitination and proteosomal degradation. These results suggest that hyperosmolarity increased the fibrosis susceptibility by phosphorylation of type II TGF-β receptors and up-regulation of the Tbeta-RI expression in MDCK cells