Smad7而非Smad6基因可抑制TGF-β诱导肾小管上皮-间充质转分化  

Smad7 instead of Smad6 blocks epithelial-mesenchymal transition induced by TGF-β in human renal proximal tubule epithelial cells

作　　者：黄云剑[1] 王梓华[1] 张静波[1] 梁莉[1] 陈枫[1] 赵景宏[1] 

机构地区：[1]第三军医大学新桥医院肾内科,重庆400037

出　　处：《细胞与分子免疫学杂志》2008年第11期1074-1078,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(30570871);重庆市自然科学基金资助项目(CSTC;2007BB5017)

摘　　要：目的:观察Smad6和Smad7基因能否抑制TGF-β诱导肾小管上皮-间充质转分化。方法:构建产生表达Smad6和Smad7基因的无辅毒腺相关病毒载体,再将病毒颗粒分别转染入人肾小管上皮细胞(HKC),细胞随机分为正常对照、TGF-β1组、Smad7组、LacZ组、TGF-β1+Smad7或Smad6组、TGF-β1+LacZ组。10μg/LTGF-β1添加入培养液孵育15、30、60、120min和3d。Western blot测定TGF-β1和Smad6或Smad7基因转染对细胞p-Smad2、E-cadherin、α-smooth muscle actin(α-SMA)表达的影响,ELISA法测定培养上清羟脯氨酸的含量的变化,倒置显微镜观察细胞形态变化。结果:与未加TGF-β1细胞相比,添加TGF-β1后15、30、60、120min胞内Smad2磷酸化水平明显增加,30min时表现最大。10μg/LTGF-β1与HKC孵育72h后,细胞α-SMA和羟脯氨酸量合成增加,E-cadherin表达减少;细胞形态变长,呈纺锤状细胞,失去鹅卵石样的形状。Smad7基因转染可抑制TGF-β1诱导Smad2磷酸化,抑制细胞α-SMA和羟脯氨酸合成,增加E-cadherin表达,维持培养细胞的上皮样形态,相反,Smad6没有这样的作用。结论:Smad7而不是Smad6基因转染能抑制TGF-β1诱导肾小管上皮-间充质转分化,这些作用可能与Smad7阻断Smad2磷酸化有关。AIM： To investigate whether Smad6 and Smad7 can regulate TGF-β-induced epithelial-mesenchymal transition in human renal proximal tubule epithelial cells. METHODS： Two recombinant adeno-associated viruses（AAV） expressing Smad6 and Smad7 genes were produced without helper virus and then they were delivered into human renal proximal tubule epithelial cells （HKCs）. The cells were randomly divided into normal controls, TGF-β1-treated group, Smad7-infected control, LacZ-infected control, TGF-β1＋Smad7 group or TGF-β1＋Smad6 group, and TGF-β1＋LacZ group. 10 μg/L of TGF-β1 was added into the cell culture at the time of 15 min, 30 min, 60 min, and 120 min and the third day. The levels of phospho-Smad2, α-smooth muscle actin （α-SMA） and E-cadherin proteins were measured by Western blot. The concentration of hydroxyproline excreted into the culture supernatant was determinated by ELISA. The morphological changes of the cells detected by inverted microscope. RESULTS： Compared with the cells in Absence of TGF-β1, the expression level of P-Smad2 in HKCs increased at 15 min, 30 min, 60 min, and 120 min with the TGF-β1 stimulation. It reached peak at 30 min. TGF-β1 treatment for 72 hours resulted in significant up-regulation of α-SMA protein expression and hydroxyproline secretion, but a marked decrease in E-cadherin expression in the culture of HKCs. 10 μg/L of TGF-β1 treatment for 72 hours also resulted in marked alteration in cell morphology. The cells lost their regular cuboidal appearance, and become elongated and spindle shaped. In the Smad7-infected cells, the overexpression of Smad7 resulted in a marked decrease of α-SMA protein and hydroxyproline synthesis, but a increase of E-cadherin protein, as well as the retainment of epithelial phenotypic appearance. These effects were associated with the inhibition of TGF-induced Smad2 activation, whereas the overexpression of Smad6 had no effect on the TGF-β-induced changes in HKCs. CONCLUSION： The overexpression of Smad7 instead of Smad6

关 键 词：SMAD7 SMAD6 上皮-间充质转分化 TGF-Β 基因治疗 

分 类 号：R392.11[医药卫生—免疫学]