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目的应用转化生长因子-β1(TGF-β1)诱导人肾小管上皮细胞-肌成纤维细胞转分化(EMT),探讨核心蛋白聚糖对人肾小管上皮细胞转分化的影响及其发生机制。方法将体外培养的人肾小管上皮细胞(HK-2)分为4组:阴性对照组;100μg/L核心蛋白聚糖组;10μg/LTGF-β1组;100μg/L核心蛋白聚糖加10μg/LTGF-β1组。应用倒置相差显微镜观察其细胞形态变化;反转录聚合酶链反应、蛋白质印迹(Western blot)法检测其波形蛋白、钙黏蛋白表达水平;Western blot法检测信号通路细胞外调节蛋白激酶(ERK)磷酸化水平;实时定量聚合酶链反应检测snail mRNA表达水平。结果1.与对照组比较,TGF-β1诱导肾小管上皮细胞从原有典型的上皮细胞形态转变为长梭形肌成纤维细胞样形态;波形蛋白和snail mRNA表达上调,钙黏蛋白表达下调,ERK的磷酸化水平增高;2.单纯核心蛋白聚糖组与对照组比较无统计学差异,而核心蛋白聚糖和TGF-β1共同刺激组与TGF-β1组比较波形蛋白和snail表达下调,钙黏蛋白表达上调,ERK磷酸水平降低。结论核心蛋白聚糖能够负性调控TGF-β1诱导EMT,核心蛋白聚糖对EMT的负性调节作用可能是通过ERK信号转导途径实现的。
Objective To investigate the effect of transforming growth factor-β1 (TGF-β1) on human renal tubular epithelial-myofibroblast transdifferentiation (EMT) and the effect of decorin on human renal tubular epithelial cell transdifferentiation. Methods Human renal tubular epithelial cells (HK-2) cultured in vitro were divided into 4 groups: negative control group, 100 μg / L decorin group, 10 μg / L TGF- β1 group, 100 μg / L decorin plus 10 μg / LTGF-β1 group. The morphological changes of cells were observed by inverted phase contrast microscope. The expression of vimentin and cadherin was detected by reverse transcriptase-polymerase chain reaction and Western blot. The expressions of extracellular regulated protein kinase (ERK) Phosphorylation level; real-time quantitative polymerase chain reaction snail mRNA expression levels detected. Results 1. Compared with the control group, TGF-β1 induced the tubular epithelial cells to transform into long spindle myofibroblast-like morphology from the typical epithelial cells. The expression of vimentin and snail mRNA was up-regulated and the expression of cadherin was down- ERK phosphorylation levels increased; 2 simple decorin group compared with the control group was no significant difference, and the core proteoglycan and TGF-β1 co-stimulation group compared with the TGF-β1 group decreased the expression of vimentin and snail, calcium Mucin expression is upregulated, ERK phosphate levels decreased. CONCLUSION: Decorin can negatively regulate TGF-β1-induced EMT. Decorin’s negative regulatory effect on EMT may be through the ERK signal transduction pathway.