热休克蛋白47对转化生长因子β_1诱导肾小管上皮细胞合成细胞外基质的影响

The effect of HSP47on the extracellular matrix synthesis induced by TGF-β_1 in the renal tubular epithelial cells

目的探讨热休克蛋白47(HSP47)对转化生长因子β1(TGF-β1)刺激的肾小管上皮细胞(HK-2)合成细胞外基质(ECM)的影响。方法以10ng/mL TGF-β1刺激HK-2细胞0、12、24、48h,蛋白印迹法检测HSP47的表达,蛋白印迹法及Real-time聚合酶链反应(PCR)法检测Ⅰ型胶原(ColⅠ)及Ⅳ型胶原(ColⅣ)的表达。然后转染HSP47小干扰RNA(siRNA)及阴性对照,后采用蛋白印迹法检测HSP47的表达,蛋白印迹法及Real-time PCR法检测ColⅠ和ColⅣ的表达。结果 TGF-β1作用于HK-2细胞后,HSP47蛋白呈时间依赖性表达上调(P<0.05),同时ColⅠ和ColⅣ蛋白及mRNA呈时间依赖性表达上调(P<0.05),转染HSP47siRNA后,蛋白印迹法显示与对照组比,TGF-β1组HSP47表达增强(P<0.01),与TGF-β1组比,HSP47siRNA组HSP47表达降低(P<0.01),对照组无明显变化(P>0.05),蛋白印记及Real-time PCR结果显示,与对照组相比,TGF-β1组ColⅠ和ColⅣ表达明显上调(P<0.01);与TGF-β1组相比,HSP47siRNA组ColⅠ和ColⅣ表达明显下调(P<0.05和P<0.01);而HSP47siRNA(-)组无明显的变化(P>0.05)。结论HSP47可促进HK-2细胞ECM合成,抑制HSP47可延缓肾间质纤维化疾病进展。

Objective To investigate the role of HSP47 on the expression of collagen Ⅰ and collage Ⅳinduced by transforming growth factorβ1（TGF-β1）.Methods HK-2cells were exposed to TGF-β1（10ng/mL）for different time（0,12,24,48h）.The expression of HSP47 was examined by Western blotting.The expressions of collagenⅠand collagen Ⅱ were examined by Western blotting and real-time PCR.Furthermore,HK-2cells were transfected with HSP47 siRNA and siRNA negative control.Then the expressions of collagenⅠand collagenⅣwere detected by Western blotting and real-time PCR,meanwhile HSP47 by Western blotting.Results Stimulation of HK-2with TGF-β1resulted in a significant increase expression of HSP47 in time-dependent（P〈0.05）.Meanwhile,TGF-β1upregulated the protein and mRNA expression of collagenⅠand collagenⅣ（P〈0.05）,all in time-dependent manner.Compared to the TGF-β1group,inhibition of HSP47 expression in HK-2downregulated the protein and mRNA expression of collagenⅠand collagen Ⅳ（P〈0.05 and P〈0.01）.However,HSP47 siRNA negative control had no significant effect on the expressions of collagenⅠand collagen Ⅳ（P〈0.05）.Conclusion HSP47 can promote ECM synthesis in HK-2cells,suppression of HSP47 can reverse the renal interstitial fibrosis.