干扰TRPM7对人肺成纤维细胞向肌成纤维细胞转化的影响

Inhibitive effect of TRPM7 on transformation of human lung fibroblasts to myofibroblasts

目的研究干扰瞬时受体电位通道7(TRPM7)对人肺成纤维细胞向肌成纤维细胞转化的影响。方法用转化生长因子-β1(TGF-β1)刺激人胚肺成纤维细胞WI-38和MRC-5,Western blot检测细胞平滑肌肌动蛋白(α-SMA)和胶原Ⅰ的表达,来评价细胞的转化。用实时聚合酶链反应(Real-time PCR)和Western blot检测细胞中TRPM7的表达。将特异性的TRPM7 si RNA转染到细胞中,并将细胞分为对照组、模型组、TRPM7 si RNA组、转染非特异性si RNA组(Scramble组)。检测转化过程中相关蛋白α-SMA、胶原Ⅰ、Smad3、p-Smad3及Smad7的表达。结果 TGF-β1(15μg/L)作用24 h后,成功诱导肺成纤维细胞转化为肌成纤维细胞。与对照组比较,模型组细胞中TRPM7 m RNA和蛋白高表达,且细胞中α-SMA、胶原Ⅰ、Smad3及p-Smad3的蛋白表达增多,Smad7表达降低,差异有统计学意义(P<0.05)。与模型组比较,TRPM7 si RNA转染组细胞TRPM7 m RNA和蛋白低表达,且α-SMA、胶原Ⅰ、Smad3及p-Smad3的表达下降,Smad7的表达升高,差异有统计学意义(P<0.05)。结论下调TRPM7可在一定程度上干预肺成纤维细胞向肌成纤维细胞转化,该作用可能与TGF-β1/Smads信号通路有关。

Objective To investigate the effects of siRNA-induced transient receptor potential channel 7 （TRPMT） on differentiation of human fetal lung fibroblasts to myofibroblasts induced by TGF-β1, providing a new target for the prevention and treatment of pulmonary fibrosis. Methods WI-38 and MRC-5 cells were cultured and stimulated with TGF-β1. The expressions of smooth muscle actin （α-SMA） and collagen I were detected by Western blot for evaluation of differentiation of fetal lung fibroblasts to myofibroblasts. The mRNA and protein expressions of TRPM7 in the cells were detected by real-time PCR and Western blot. The cells were divided into control group, model group, nonspecific transfected group and TRPM7 siRNA group. Western blot was performed to detect the expressions of α-SMA, collagen I , Smad3, p-Smad3 and Smad7, respectively. Results WI-38 and MRC-5 cells were successfully transformed into muscle fibroblasts in the presence of TGF-β1 （15 μg/L） for 24 h. Compared with the control group, the up-regulation of TRPM7, α- SMA, collagen I, Smad3 and p-Smad3, and the down-regulation of Smad7 were observed in the model group （P 〈 0.05）. Furthermore, the expressions of TRPM7, α-SMA, collagen I , Smad3 and p-Smad3 were remarkably down-regulated in the TRPM7 siRNA group compared with the model group （P 〈 0.05）, while TRPM7 siRNA significantly increased Smad7 level compared with the model group （P 〈 0.05）. Conclusions TRPM7 siRNA could restrain differentiation of fetal lung fibroblasts to myofibroblasts to some extent, which may be related to the activation of TGF-β1/Smads signal pathway.