RNA干扰抑制肾小管上皮细胞TSP1表达和TGF-β1活化  

RNA Interference Targeting Thrombospondin 1 Suppresses the Activation of Latent Transforming Growth Factor-β1 in Human Renal Tubular Epithelial Cell

作　　者：吴开胤[1] 王伟铭[1] 黄秋花 周同[1] 陈楠[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院肾脏科 [2]上海血液病研究所,上海200025

出　　处：《细胞生物学杂志》2006年第2期206-212,共7页Chinese Journal of Cell Biology

基　　金：国家自然科学基金资助项目(No.30270613);上海市重点学科(No.T0201);上海市卫生局重点学科基金(No.05Ⅲ001);上海市卫生局重点项目(No.2003ZD002)~~

摘　　要：血小板反应蛋白1(TSP1)是转化生长因子-β1(TGF-β1)体内重要的活化因子,而后者又是致肾小管间质纤维化的关键因素。观察了针对TSP1的小双链干扰RNA(siRNA-TSP1),抑制由血管紧张素II(AngII)诱导的肾小管上皮细胞TGF-β1过度活化。将根据人TSP1基因序列设计的特异siRNA-TSP1转染人肾小管上皮细胞系(HK-2),利用Western印迹、RT-PCR、流式细胞仪及ELISA等方法,检测了TSP1、TGF-β1及其信号蛋白Smad2与p-Smad2、纤维连接蛋白(FN)和纤溶酶原激活剂抑制物-1(PAI-1)的基因转录水平、蛋白质表达或蛋白质活性。结果显示,siRNA-TSP1能有效转染HK-2细胞,并以剂量依赖方式显著抑制TSP1的基因转录与合成;其对TGF-β1的合成影响较小,但能明显抑制TGF-β1的活化。此外siRNA-TSP1可阻抑TGF-β1依赖的Smad2磷酸化,减少细胞外基质FN以及PAI-1的合成。研究结果提示,由于TSP1是TGF-β1重要的内源性活化因子,故针对TSP1的RNA干扰能在体外有效抑制TSP1表达并相应调抑了TGF-β1的活化。Thrombospondin 1 （TSP1） is a major endogenous activator for transforming growth factor-β1 （TGF-β1）, which plays an critical role on the development of renal tubulointerstitial fibrosis. The purpose of the study was to determine whether small interference RNAs （siRNA） targeting the thrombospondin-1 could be used to suppress the over-activation of TGF-β1 induced by angiotensin Ⅱ in human renal tubular epithelial cells. TSP1 specific siRNA designed from the human gene sequence was transfected into cultured human renal tubular epithelial cells （HK-2）. The protein and transcript levels of the TSP1 and TGF-β1 were determined by Western bloting and RT-PCR. The co-localization of TSP1 and TGF-β1 was observed by flow-cytometry. Western blotting was performed to measure the level of Smad2 and phsophorylated-Smad2. The secreting extracellular matrix such as fibronectin and PAI-1 was examined by ELISA. TGF-β1 bioactivity was determined by ELISA. siRNA targeting TSP1 was successfully transferred into HK-2 cells and markedly inhibited de novo synthesis of TSP1 in a dose dependent manner. This effect was accompanied by decreased activation TGF-β1 but the total level of TGF-β1 remained unaffected, siRNA additionally inhibited TGF-β1-dependent Smad-signaling pathway, markedly suppressed accumulation of fibronectin as well as transcription of TGF-β1 target gene PAI-1. TSP1 is the major endogenous activator of TGF-β1, TSP1 specific siRNA were efficacious in vitro knocking down the expression of TSP1 and further suppressing TGF-β1 activation.

关 键 词：RNA干扰 血小板反应蛋白1 转化生长因子-Β1 肾小管上皮细胞 肾纤维化 

分 类 号：R692.6[医药卫生—泌尿科学]