摘要
目的观察转化生长因子（TGF）β1诱导的正常人近端肾小管上皮细胞（HK-2）转分化（EMT）过程中黏着斑激酶（FAK）的表达及下调FAK的表达后对TGF-β1诱导的HK-2细胞转分化进程的影响。方法应用TGF-β1（10Ixg／L）刺激HK-2细胞，采用RT—PCR、Western印迹和免疫荧光方法分别检测E钙黏蛋白（E—cadherin）、α平滑肌肌动蛋白（α-SMA）、FAK mRNA和蛋白的表达及磷酸化（p）-FAK（Tyr397）的蛋白表达。应用Lipofectmine2000将FAK siRNA转染HK-2细胞，采用Western印迹观察下调表达FAK对上述指标的影响。结果TGF—β1刺激后，HK-2细胞α-SMA蛋白和mRNA水平上调，E—cadherin蛋白和mRNA表达下调。FAK蛋白和mRNA随时间的延长表达逐渐增多，48h达到高峰。p-FAK（Tyr397）蛋白表达趋势与FAK相同。脂质体转染siRNA后FAK的mRNA和蛋白分别下调了50％和41％，下调表达FAK后可以显著抑制TGF-β1诱导的HK-2细胞α—SMA蛋白的上调表达，逆转E—cadherin蛋白的下调表达。结论在TGF—β1诱导的HK-2细胞转分化进程FAK蛋白表达上调，敲低FAK蛋白表达后可以部分减轻EMT的程度，提示FAK在TGF-β1诱导的肾小管上皮细胞转分化和肾脏纤维化中发挥一定的作用。
Objective To investigate the expression of focal adhesion kinase （FAK） in epithelial-mesenchymal transition （EMT） of TGF-β1-stimulated HK-2 cells and the effect of FAK knockdown by small interfering RNA on EMT. Methods HK-2 cells were grown in DMEM- F12 medium supplemented by 10% fetal bovine serum （FBS）. HK-2 cells were cultured in free serum medium for 24 h, then were stimulated by TGF-β1（10 μg/L）. The expression of E-cadherin, α-SMA, FAK mRNA and protein were detected by RT-PCR, Western blot and immunofluorescenee, respectively. The expression level of pbospborylated FAK-Tyr397 was detected by Western blot. HK-2 cells were transfeeted with 200 nmol/L FAK-siRNA or negative control siRNA using Lipofectamine 2000. Then the expression of E-cadherin, α-SMA, FAK protein was detected by Western blot. Results The expression of E-cadherin mRNA and protein was markedly decreased in HK-2 cells induced by TGF-β1, and the expression of α-SMA mRNA and protein was dramatically increased. Western blot analysis demonstrated that then protein levels of FAK and p-FAK （Tyr397） were progressively increased in a time-dependent manner in response to TGF-β1 treatment in HK-2 cells. When transfected with FAK-siRNA, the FAK mRNA and proteinexpression was markedly inhibited with 50% and 41%. Knockdown expression of FAK led to a severe blockage of TGF-β1-induced E-cadherin suppression and α-SMA induction. Conclusions The expression of FAK is up-regulated in HK-2 cells stimulated by TGF-β1. But the EMT induced by TGF-β1 in HK-2 cells is inhibited by FAK knockdown, which suggests that FAK plays an important role in TGF-β1-indueed tubular EMT and renal fibrosis.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2009年第10期771-775,共5页
Chinese Journal of Nephrology
关键词
转化生长因子
黏着斑激酶
上皮细胞转分化
Transforming growth factor beta
Focal adhesion kinase
Epithelial- mesenchymal transition
