CAJ | 학술논문

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(10 μg/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,48 h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.
목적 관찰전화생장인자(TGF)β1유도적정상인근단신소관상피세포(HK-2)전분화(EMT)과정중점착반격매(FAK)적표체급하조FAK적표체후대TGF-β1유도적HK-2세포전분화진정적영향.방법 응용TGF-β1(10 μg/L)자격HK-2세포,채용RT-PCR、Western인적화면역형광방법분별검측E개점단백(E-cadherin)、α평활기기동단백(α-SMA)、FAK mRNA화단백적표체급린산화(p)-FAK(Tyr397)적단백표체.응용Lipofectmine2000장FAK siRNA전염HK-2세포,채용Western인적관찰하조표체FAK대상술지표적영향.결과 TGF-β1자격후,HK-2세포α-SMA단백화mRNA수평상조,E-cadherin단백화mRNA표체하조.FAK단백화mRNA수시간적연장표체축점증다,48 h체도고봉.p-FAK(Tyr397)단백표체추세여FAK상동.지질체전염siRNA후FAK적mRNA화단백분별하조료50%화41%,하조표체FAK후가이현저억제TGF-β1유도적HK-2세포α-SMA단백적상조표체,역전E-cadherin단백적하조표체.결론 재TGF-β1유도적HK-2세포전분화진정FAK단백표체상조,고저FAK단백표체후가이부분감경EMT적정도,제시FAK재TGF-β1유도적신소관상피세포전분화화신장섬유화중발휘일정적작용.
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 immunofluorescence,respectively.The expression level of phosphorylated FAK-Tyr397 was detected by Western blot.HK-2 cells were transfected 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 protein expression 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-induced tubular EMT and renal fibrosis.