单核细胞趋化蛋白-1诱导肾小管上皮-肌成纤维细胞转化的作用及机制探讨

Effects of induction of tubular epithelial-myofibroblast transition by monocyte chemoattractant protein-1 and mechanism thereof： an in vitro experiment

目的探讨单核细胞趋化蛋白（MCP）-1诱导肾小管上皮细胞发生上皮-肌成纤维细胞转化（EMT）的作用和细胞分子机制，主要是EMT过程中Erk1／2、P38MAPK、RhoA、RhoC、Snail表达水平的变化。方法将HK-2细胞分为阴性对照组，阳性对照组（TGF-β1、5μg／L），MCP-1作用组（0．1、1、10、100μg／L），观察不同时间（12、24、48、72h）MCP-1刺激与α-SMA表达水平之间的变化。用Western印迹方法检测肾小管上皮细胞PErk1／2、Erk1／2、P-P38MAPK、P38MAPK、RhoA蛋白水平的变化，用逆转录-聚合酶链反应（RT-PCR）检测RhoC、Snail基因水平的变化。此外，分别观察Erk、P38MAPK、Rho信号通路的阻断剂（PD98059、SB203580、Y27632）对MCP-1诱导EMT的影响。结果在不同浓度MCP-1刺激下，HK-2细胞α-SMA mRNA及蛋白表达水平均明显增强，以1μg／L刺激下表达最强；在1μg／LMCP-1作用下，24、48、72hα-SMAm RNA及蛋白表均达显著高于阴性对照组（均P〈0．05），48h作用达到最高峰。在不同浓度（0．1、1、10、100μg／L）MCP-1刺激5min时，HK-2细胞（PErk1／2）／（Erk1／2）和（P-P38MAPK）／（P38MAPK）的比值均显著高于阴性对照组（均P〈0．05），并表现为剂量依赖性。不同浓度MCP-1刺激下RhoA蛋白水平和RhoC基因表达水平与阴性对照组比较差异均无统计学意义（均P〉0．05）。PD98059可以部分阻断MCP-1诱导的肾小管上皮细胞α-SMA蛋白表达水平，与阳性对照组相比，差异有统计学意义（P〈0．05）；而SB203580及Y27632均不能阻断MCP-1诱导的α-SMA蛋白表达，与阳性对照组比较差异无统计学意义（均P〉0．05）。MCP-1（10、100μL）刺激下Snail基因表达水平明显升高，与阴性对照组相比差异有统计学意义（P〈0．05）。结论MCP-1诱导体外培养的HK-2细胞发生EMT与上调Erk1／2信号转导通路有关，并可能与上调Snail转录因子水平有关，本研究未能证实该机制与P38MAPK信号通路及Rho信号转导通路有关。

Objective To examine the effects of monocyte chemoattractant protein-1 （MCP-1） on epithelial-myofibroblast transition （EMT） of renal proximal tubular epithelial cells and the possible mechanism involved. Methods Human renal proximal tubular epithelial cells of the line HK-2 were cultured and divided into three groups： negative control group; positive control group, treated with transforming growth factor （TGF）-β1 5 μg/L, and MPC-1 group, treated with MCP-1 0.1, 1, 10, and 100 μg/L for 24 h, 36 h, 48 h, and 72 h respectively. The expressions of α-smooth muscle actin （α-SMA） mRNA and protein of cells were detected by s. Western blotting and RT-PCR were used to detect the expression of P-Erk1/2, Erk1/2, P-P38MAPK, P38MAPK, and RhoA protein levels of the HK-2 cells, and RT-PCR was used to detect the expression of RhoC and Snail mRNA. Specific inhibitors of the Erk, P38MAPK, and Rho signal transduction pathways PD98059, SB203580, and Y27632 were added into the culture fluid of HK-2 cells respectively, 1 h later MCP-1 μg/L was added for 48 h, and Western blotting was used to detect the protein expression of α-small muscle actin （SMA）. Results The expression levels of α- SMA protein and mRNA significantly increased in the MCP-1 treated HK-2 cells, and these expression levels were the highest in the HK-2 cells treated with MCP-1 1 μg/L for 48 h. The ratios of （P-Erk1/2）/ （Erk1/ 2） and P-P38MAPK/P38MAPK were significantly increased （ all P 〈 0.05 ） in the MCP-1 treated HK-2 cells with the highest ratios seen in the HK-2 cells treated by 100 μg/L of MCP-1. The expression levels of RhoA protein and RhoC mRNA of the HK-2 cells stimulated with MCP-1 of different concentrations were not significantly different （all P 〉 0.05 ）. MCP-1 induced expression of a-SMA was only partly inhibited by PD98059 but not by SB203587 or Y27632. MCP-1 dose-dependently increased the expression of Snail mRNA of the HK-2 cells compared with those of the negative control cells. The level of Snail mRNA was the highest in the HK-2 cells treated with 100 μg/L MCP-1. Conclusion MCP-1 may induce the EMT of renal proximal tubular epithelial cells in vitro, and this effect may involve upregulation of Erk1/2 Map kinase signal pathways and Snail mRNA expression. P38MAPK or Rho kinase signal pathways can not be proven to be involved in MCP-1 induced EMT.