CAJ | 학술논문

目的阐明热休克蛋白47(HSP47)在转化生长因子β1(TGF-β1)刺激的人近端肾小管上皮细胞(HK-2细胞)向间充质细胞转分化(EMT)中的作用及可能机制,探讨以HSP47为靶点抑制TGF-β1诱导的HK-2细胞EMT过程.方法 Western印迹检测不同浓度TGF-β1(0、2.5、5、10 μg/L)刺激HK-2细胞不同时间(0、12、24、48 h)后HSP47的表达;用10μg/L TGF-β1刺激HK-2细胞,Western印迹及实时定量PCR(real-time PCR)检测波形蛋白(vimentin)及紧密连接蛋白(zona occludens-1,ZO-1)的表达变化,Western印迹检测不同时间磷酸化(p)-Smad3及Smad3的表达;转染HSP47 siRNA及HSP47 siRNA阴性对照,对照组为无基因抑制效果的一段siRNA,再予TGF-β1刺激,Western印迹检测HSP47、p-Smad3及Smad3的表达,Western印迹及real-time PCR检测vimentin及ZO-1的表达.结果 TGF-β1干预HK-2细胞,HSP47蛋白呈浓度和时间依赖性表达上调(均P＜0.05).Western印迹、real-time PCR结果显示10 μg/L TGF-β1刺激后,HK-2细胞vimentin蛋白及mRNA表达呈时间依赖性升高(均P＜0.05),ZO-1蛋白及mRNA表达呈时间依赖性降低(均P＜0.05).Western印迹结果显示TGF-β1促进Smad3磷酸化,上调p-Smad3/Smad3表达(P＜0.05),在30 min时达到峰值,随后稍下降,在24～ 48 h达到另一峰值.与TGF-β1组相比,转染HSP47 siRNA后HSP47蛋白表达明显下调(P＜0.05),同时ZO-1蛋白及mRNA表达上调(均P＜0.05),vimentin蛋白及mRNA表达下调(均P＜ 0.05);同时p-Smad3/Smad3表达下调(P＜0.05).结论 HSP47可促进肾小管上皮细胞EMT,可能与TGF-β1-Smad3信号通路活化有关.
목적 천명열휴극단백47(HSP47)재전화생장인자β1(TGF-β1)자격적인근단신소관상피세포(HK-2세포)향간충질세포전분화(EMT)중적작용급가능궤제,탐토이HSP47위파점억제TGF-β1유도적HK-2세포EMT과정.방법 Western인적검측불동농도TGF-β1(0、2.5、5、10 μg/L)자격HK-2세포불동시간(0、12、24、48 h)후HSP47적표체;용10μg/L TGF-β1자격HK-2세포,Western인적급실시정량PCR(real-time PCR)검측파형단백(vimentin)급긴밀련접단백(zona occludens-1,ZO-1)적표체변화,Western인적검측불동시간린산화(p)-Smad3급Smad3적표체;전염HSP47 siRNA급HSP47 siRNA음성대조,대조조위무기인억제효과적일단siRNA,재여TGF-β1자격,Western인적검측HSP47、p-Smad3급Smad3적표체,Western인적급real-time PCR검측vimentin급ZO-1적표체.결과 TGF-β1간예HK-2세포,HSP47단백정농도화시간의뢰성표체상조(균P＜0.05).Western인적、real-time PCR결과현시10 μg/L TGF-β1자격후,HK-2세포vimentin단백급mRNA표체정시간의뢰성승고(균P＜0.05),ZO-1단백급mRNA표체정시간의뢰성강저(균P＜0.05).Western인적결과현시TGF-β1촉진Smad3린산화,상조p-Smad3/Smad3표체(P＜0.05),재30 min시체도봉치,수후초하강,재24～ 48 h체도령일봉치.여TGF-β1조상비,전염HSP47 siRNA후HSP47단백표체명현하조(P＜0.05),동시ZO-1단백급mRNA표체상조(균P＜0.05),vimentin단백급mRNA표체하조(균P＜ 0.05);동시p-Smad3/Smad3표체하조(P＜0.05).결론 HSP47가촉진신소관상피세포EMT,가능여TGF-β1-Smad3신호통로활화유관.
Objective To detect the expression of heat shock protein 47(HSP47) in renal proximal epithelial cell lines (HK-2) and to investigate the role of HSP47 in the progress of transforming growth factor β1 (TGF-β1) induced epithelial-mesenchymal transdifferentiation (EMT) in HK-2 cells.Methods HK-2 cells were exposed to TGF-β1 (0,2.5,5,10 μg/L) for different time (0,12,24,48 h).The expression of HSP47 was examined by Western blotting.Then HK-2 cells were exposed to 10 μg/L TGF-β1,the expressions of vimentin,zona occludens-1 (ZO-1) were examined by Western blotting and real-time PCR.Furthermore,the expressions of p-Smad3 and Smad3 were examined by Western blotting.HK-2 cells were transfected with HSP47 siRNA and siRNA negative control before exposing to TGF-β1.Then the expressions of vimentin,ZO-1 were detected by Western blotting and real-time PCR,meanwhile Western blotting for HSP47,p-Smad3 and Smad3.Results Stimulating HK-2 with TGF-β1 resulted in a significant increased expression of HSP47 in time-and concentration-dependent manner (P ＜ 0.05).Meanwhile,TGF-β1 up-regulated the protein and mRNA expression of vimentin (P ＜ 0.05),and down-regulated the protein and mRNA expression of ZO-1 (P ＜ 0.05),all in time-dependent manner.Stimulating HK-2 with TGF-β1 resulted in phosphorylation of Smad3,which was peaked at 30 min,slightly decreased at 1 h,and then increased again between 24 and 48 h (P ＜ 0.05).Compared to the TGF-β1 group,inhibition of HSP4.7 expression in HK-2up-regulated the protein and mRNA expression of ZO-1,down-regulated the protein and mRNA expression of vimentin (P ＜ 0.05) and down-regulated the ratio of p-Smad3/Smad3.HSP47 siRNA negative control had no significant effect on the expressions of ZO-1,vimentin and p-Smad3/Smad3 (P ＞ 0.05).Conclusion HSP47 can promote the EMT of renal tubular epithelial cell which is possibly via the TGF-β1-Smad3 pathway.