RhoA／Rho激酶信号通路在TGF—β1诱导的人皮肤成纤维细胞表型转化中的作用

The effect of RhoA/Rho kinase signal pathway on TGF-β-induced phenotypic differentiation of human dermal fibroblasts

目的 观察TGF—β1对人皮肤成纤维细胞表型转化的作用是否受到RhoA／Rho激酶信号通路的影响，以探究该通路是否参与了人皮肤成纤维细胞的表型转化。方法原代培养人皮肤成纤维细胞，将第4代细胞用TGF—β1（10ng／ml）刺激后，分不同作用时间（0、3、6、24h）提取细胞内总蛋白。BCA法测定总蛋白浓度，Western—blot检测a—SMA的表达水平；并用相同的方法检测不同浓度TGF—β1（0、2、10、50ng／ml）刺激人皮肤成纤维细胞后，a-平滑肌肌动蛋白（d—smoothmuscleactin，a—SMA）的表达水平。用RhoA／Rho激酶信号通路阻断剂Y-27632处理后，再用TGF—β1刺激，作为实验组，将未作处理的正常细胞作为空白对照组，Y-27632（10μmol／L）组作为阴性对照组，只用TGF-β（10ng／ml）刺激组作为阳性对照组，分别检测a-SMA的表达差异。使用ANOVA对蛋白表达量进行统计学分析，P〈0．05为差异有统计学意义。结果10ng／mlTGF-β按不同作用时间刺激人皮肤成纤维细胞后，a—SMA的表达量分别为：0h为1．0，3h为1．9±0．2、6h为2．1±0．1，24h为3．1±0．1。24h较其他3个时间点a-SMA表达量明显上升（n=4，P〈0．05）。不同浓度TGF—β1刺激人皮肤成纤维细胞后，a—SMA的表达量分别为：0ng／ml为1．0，2ng／ml为1．4±0．2，10ng／ml为3．2±0．1，50ng／ml为3．1±0．2。10ng／ml表达量明显高于0ng／ml和2ng／ml（n=4，P〈0．05），较50ng／ml差异无统计学意义（n=4，P〉0．05）。用Y-27632（10μmol／L）处理后，再用TGF—β1刺激，细胞的表型转化明显受到抑制，实验组a—SMA的表达量（1．2±0．2）较阳性对照组（2．9±0．1）明显降低（n=5，P〈0．05），与空白对照组（1．0）和阴性对照组（1．1±0．1）相比差异均无统计学意义（n=5，P〉0．05）。结论RhoA／Rho激酶信号通路参与了TGF—B。诱导的人皮肤成纤维细胞表型转化过程。

Objective To examine the effect of RhoA/Rho kinase signal pathway on TGF-β1 induced phenotypic differentiation of human dermal fibroblasts. Methods The 4th generation of primary cultured human dermal fibroblasts were stimulated with TGF-β1 （ 10 ng/ml）. The expression of a-SMA was detected after treatment with TGF-β1 for 0, 3, 6, and 24 h. The expression of a-SMA was also detected after treatment with different concentration of TGF-β1 （0, 2, 10, 50 ng/ml）. Then the human dermal fibroblasts （4th generation）were stimulated with TGF-β1 （10 ng/ml） after being treated with the RhoA/Rho kinase signaling pathway inhibitor Y-27632 （10umol/ml）. The fibroblasts were treated with nothing as sham control, or with Y-27632 （10umol/L） only as negative control group, or with TGF-β1 （10 ng/ml） only as positive control group. The expression of a-SMA was detected in all the groups. Protein expression was analyzed with ANOVA statistical method. Results a-SMA expression in fibroblasts with 10 ng/ml TGF-β1 stimulation forO, 3, 6, 24 h was 1.0, 1.9 ±0.2, 2.1 ±0.1, 3.1 ±0.1, respectively, a-SMA expression in 24 h group was significantly higher than that in other three groups （n= 4, P 〈 0. 05）. a-SM expression in human dermal fihroblasts after stimulation with different concentration of TGF-β1 （ 0, 2, 10, 50 ng/ml） was 1.0, 1.4±0.2, 3.2 ±0. 1, 3.1 ± 0.2, respectively, a-SMA expression in 10 ng/ ml group was significantly higher than that in 2 ng/ml group and control group （ n = 4, P 〈 0.05 ）. There was no statistical difference in a-SMA expression between 10 ng/ml group and 50 ng/m] group （n = 4, P 〉 0. 05）. With both Y-27632 （10 μmol/L） and TGF-β1 stimulation, the cell phenotype differentiation was inhibited, a-SMA expression in experimental group （ 1.2 ± 0.2） was significantly reduced, when compared with that in positive control group （2.9 ± 0. 1 ） （ n = 5, P 〈 0. 05 ）. There was no significant difference （n = 5 ,P 〉 0. 05） in a-SMA expression between control group （1.0） and negative control group （ 1.1 ± 0. 1）. Conclusions RhoA/Rho kinase signaling pathway should be involved in TGF-β1-induced phenotypic differentiation of human dermal fibrohlasts.