核心蛋白聚糖抑制转化生长因子β1诱导的人肾小管上皮细胞转分化机制研究

摘要

目的 研究核心蛋白聚糖抑制转化生长因子β1(TGF-β1)诱导的人肾小管上皮细胞-间充质细胞-转分化(EMT)的机制.方法 将体外培养的人肾小管上皮细胞(HK-2)分为4组:(1)阴性对照组;(2)100 ng/ml核心蛋白聚糖组;(3)10 ng/ml TGF-β1组;(4)100 ng/ml核心蛋白聚糖和10ng/ml TGF-β1组.Western blot法检测信号通路ERK、PI3K、Smad_3的磷酸化水平和β-catenin的蛋白水平;实时定量-PCR检测snail mRNA的表达情况,逆转录-PCR检测淋巴细胞增长因子1(LEF-1)mRNA的表达情况.结果 (1)与对照组相比,TGF-β1诱导组ERK(1.11 ±0.09:0.47 ±0.07)、PI3K(14.79 ±1.02:2.48 ±0.06)、Smad_3(0.95 ±0.02:0.08 ±0.01)的磷酸化水平明显增高,snail(2.59±0.70:1.02±0.13)、LEF-1(1.85 ±0.08:0.30 ±0.11)的mRNA的表达量明显增高,β-catenin(1.46 ±0.20:0.49±0.05)的最明显增高;(2)单纯核心蛋白聚糖组与对照组相比,所得结果 差异均无统计学意义.核心蛋白聚糖和TGF-β1共同刺激组与TGF-β1组相比,ERK(0.58 ±0.08)的磷酸化水平及snail mRNA(1.24 ±0.03)的表达量明显降低而PI3K(15.84 ±1.64),Smad_3(0.90 ±0.04)的磷酸化水平,LEF-1的mRNA(1.64 ±0.07)、β-catenin(1.42±0.09)的量差异无统计学意义.结论 核心蛋白聚糖抑制TGF-β1诱导的人肾小管上皮细胞EMT可能是通过阻止ERK信号转导通路实现的.%Objective To investigate the mechanisms of decorin inhibiting epithelial-to-mesenchymal transition (EMT)induced by transforming growth factor β1(TGF-β1)in renal tubular epithelial cells.Method HK-2 cells in vitro were divided into 4 groups:(1)negative control group;(2) decorin group,added with decorin 100 ng/ml;(3)TGF-β1 group,added with TGF-β1 10 ng/ml;(4) decorin and TGF-β1 group,added with decorin 100 ng/ml and TGF-β1 10 ng/ml.The protein level of phosphor-ERK,phosphor-PI3K,phosphor-Smad_3 and β-catenin was detected by Western blotting method.The snail mRNA level was tested by real time-PCR,while the lymphoid enhancer factor-1(LEF-1)mRNA level was measured by RT-PCR.Results The snail(2.59 ±0.70:1.02 ±0.13)and LEF-1 mRNA(1.85±0.08:0.30±0.11)were significantly up-regulated,meanwhile the protein level of phosphor-ERK(1.11±0.09:0.47±0.07).phosphor-P13K(14.79±1.02:2.48±0.06),phosphor-Smad_3(0.95±0.02:0.08 ±0.01)and β-catenin(1.46 ±0.20:0.49 ±0.05)were significantly increased in TGF-β1 group compared to control group,while there were no statistically significant difference in all figures between control group and decorin group.The phosphor-ERK protein level(0.58 ±0.08)and the snail mRNA level(1.24 ±0.03) were significantly down-regulated in TGF-β1 and decorin group compared to TGF-β1 group,however there were no statistically significant differences in the level of phosphor-PI3K(15.84 ±1.64),phosphor-Smad_3 (0.90 ±0.04)and β-catenin(1.42 ±0.09)between these two groups.Conclusion Decorin inhibited EMT induced by TGF-β1 which may be through blocking the ERK signal transduction pathway.