结缔组织生长因子诱导人肾小管上皮细胞向间充质细胞转化的实验研究

Inducing of epithelial mesenchymal transition of HK-2 cells by connective tissue growth factor in vitro

目的探讨结缔组织生长因子（CTGF）对肾小管上皮细胞向间充质细胞转化的影响。方法体外培养的人肾小管上皮细胞（HK-2），按是否用CTGF处理将其分为实验组和对照组。直接免疫荧光法、间接免疫化学检测培养72h后肾小管上皮细胞E—cadherin、波形蛋白、α-平滑肌肌动蛋白（α—SMA）、细胞外信号调节激酶2（ERK2）的表达；Western blot方法在72h检测E—cadherin、波形蛋白和ERK2的表达；Boyden小室在1、3、5d检测细胞迁移能力的变化。结果（1）实验组上皮细胞表型标志蛋白E-cadherin随着时间的延长而表达递减，波形蛋白表达渐增，ERK2表达上调；（2）在第1天，实验组与对照组细胞迁移能力差异无统计学意义，第3天实验组迁移至滤膜下面细胞多于对照组，第5天实验组细胞迁移数明显高于对照组[（45．0±1．1）：（14．0±1．2），P〈0．05]。结论在体外，人肾小管上皮细胞由CTGF刺激表现出向间充质细胞转化的特性，并且ERK2信号转导通路可能参与了对这一过程的调控效应。

Objective To investigate the role of connective tissue growth factor （CTGF） in epithelial mesenchymal transition of HK-2 cells in vitro. Methods HK-2 ceils were randomly divided into two groups： （ 1 ） control group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum only; and （2） experimental group including ceils cultured in DMEM medium supplemented with 10% fetal bovine serum and recombinant CTGF at a final concentration of 5 μg/L. The cells were collected at 72 h time points. Direct immunofluorescence staining and immunohistochemistry were used to evaluate the E-cadherin, Vimentin, α-SMA and ERK2 in cells. Western-blotting was used to detect the E-cadherin, Vimentin and ERK2 protein expression. Boyden Chamber was used to detect the migration of tubular endothelium at 1 d, 3 d and 5 d. Results There were less E-cadherin but more Vimentin expressed in cells of the experimental group. The presence of α-SMA was detected at 48 h with peak at 72 h in the cells of the experimental group. On the first day, the cellular migration in the two groups showed no difference. However, after 3 days, the transformed cells migrated surpassed the control group with peak at the5th day [（45.0±1.1）： （14.0±1.2）, P〈0.05）]. Conclusion Connective tissue growth factor induces mesenchymal transformation of HK-2 cells, in which the ERK2 signaling pathway may play an important role.