CAJ | 학술논문

目的探讨在转化生长因子(TGF)β1致大鼠肾小管上皮细胞(NRK52E)发生上皮细胞向间质细胞转分化(EMT)过程中囊泡型H+-ATP酶(V-ATPase)B亚基的变化及其可能意义.方法 NRK52E细胞无血清培养后予TGF-β1(10 μg/L)刺激不同时间(0、6、12、24、48、72 h),应用实时定量PCR、Western印迹、免疫荧光技术检测α平滑肌肌动蛋白(α-SMA)、E钙黏素(E-cadhefin)、V-ATPase B亚基(B1、B2)mRNA、蛋白表达及分布的变化.结果 TGF-β1刺激NRK52E细胞48 h后α-SMA mRNA及蛋白表达显著上调,E-cadherin mRNA及蛋白表达显著下调,同时V-ATPase B2亚基mRNA及蛋白表达也显著增加(均P<0.05).但是B1亚基在细胞内表达很低,刺激后也未见显著变化.免疫荧光也显示V-ATPase B2亚基在细胞内的分布明显增加并向胞膜聚集.结论在NRK52E内主要分布的是V-ATPase B2亚基.TGF-B1刺激NRK52E EMT过程中V-ATPase B2亚基表达显著增加,这提示B2亚基可能参与肾小管EMT过程.
목적 탐토재전화생장인자(TGF)β1치대서신소관상피세포(NRK52E)발생상피세포향간질세포전분화(EMT)과정중낭포형H+-ATP매(V-ATPase)B아기적변화급기가능의의.방법 NRK52E세포무혈청배양후여TGF-β1(10 μg/L)자격불동시간(0、6、12、24、48、72 h),응용실시정량PCR、Western인적、면역형광기술검측α평활기기동단백(α-SMA)、E개점소(E-cadhefin)、V-ATPase B아기(B1、B2)mRNA、단백표체급분포적변화.결과 TGF-β1자격NRK52E세포48 h후α-SMA mRNA급단백표체현저상조,E-cadherin mRNA급단백표체현저하조,동시V-ATPase B2아기mRNA급단백표체야현저증가(균P<0.05).단시B1아기재세포내표체흔저,자격후야미견현저변화.면역형광야현시V-ATPase B2아기재세포내적분포명현증가병향포막취집.결론 재NRK52E내주요분포적시V-ATPase B2아기.TGF-B1자격NRK52E EMT과정중V-ATPase B2아기표체현저증가,저제시B2아기가능삼여신소관EMT과정.
Objecfive To investigate the change of V-ATPase B subunits on epithelial to mesenchymal transition (EMT)in rat renal tubular epithelial cells (NRK52E) stimulated by transforming growth factor β1 (TGF-β1). Methods NRK52E cells were stimulated by TGF-β1 (10 μg/L)for O h(control),12 h,24 h,48 h,72 h after sefrum-free culture for 24 h.The mRNA and protein expression of E-cadherin,α-SMA,B2 and B1 subunits of V-ATPase were detected by real-time PCR,Western blotting and immunofluorescence. Results After stimulated by TGF-β1 (10 μg/L)for 48 h,the expression of α-SMA was markedly increased(P<0.05),but the expression of E-cadherin was dramatically decreased(P<0.05).Meanwhile,the expressions of V-ATPase subunit B2 was significantly increased (P<0.05).However,the B1 subunit distributed rarely in NRK 52E cells,and did not increase after TGF-β1 stimulation.Double-label immunofluoerscence staining also showed that the V-ATPase B2 subunit was increased in the cytosol.tending to accumulate to the cell membrane after TGF-β1 stimulation. Conclusions The main isoform of V-ATPase distributed in NRK52E cells is B2 subunit.B2 subunit is increased alone with TGF-β1-induced EMT.It may suggest that V-ATPase B2 subunit may play a potential role in TGF-β1-induced tubular EMT and renal fibrosis.