吗替麦考酚酯对肺成纤维细胞的转化及功能影响

Effects of mycophenolate mofetil on transdifferentiation and function of lung fibroblasts

目的:通过吗替麦考酚酯(MMF)对体外培养肺成纤维细胞(LF)转化为肌成纤维细胞(MF)过程的干预,观察MMF对LF转化及CTGF、FN蛋白表达的影响。方法:体外培养新生大鼠来源的LF,加入转化生长因子-β1(TGF-β1)诱导转化为MF,分别加入终浓度为0μmol/L(对照组)、0.1μmol/L(低浓度组)、1μmol/L(中浓度组)、10μmol/L(高浓度组)的MMF进行干预;倒置显微镜下观察LF和MF形态,并用细胞免疫荧光的方法鉴定波形蛋白和α-平滑肌肌动蛋白(α-SMA),分析MMF对LF转化的影响;ELISA方法检测结缔组织生长因子(CTGF)和纤维连接蛋白(FN)的水平。结果:LF经TGF-β1诱导96 h后转化成MF,细胞免疫荧光结果显示MMF能够抑制α-SMA的表达,但对已分化的肌成纤维细胞表型变化无影响;ELISA结果显示与对照组比较,MMF组CTGF和FN分泌水平均显著下降(P<0.05),并呈一定的浓度依赖。结论:MMF能够抑制肺成纤维细胞转化和CTGF、FN表达,提示MMF有抗纤维化作用,影响CTGF和FN表达可能是作用途径之一。

Objective：To investigate the effects of mycophenolate mofetil （ MMF） on the differentiation and connective tissue growth factor（CTGF）and fibronectin（FN）expression of lung fibroblasts（LF）through interfering the transdifferentiation of LF into MF in vitro.Methods：LF of neonatal rat were cultured in vitro ,induced into MF by transforming growth factor-β1（TGF-β1）,and treated with different concentrations of MMF ,which was 0μmol/L（control group）,0.1μmol/L（low dose group）,1μmol/L（middle dose group）or 10 μmol/L（ high dose group ） .Morphology of LF and MF were observed by inverted phase contrast microscope , the expressions of vimentin and α-smooth muscle actin （α-SMA） were identified by immunofluorescence staining ,and then analyzed the effect of MMF on the transdifferentiation of fibroblasts .ELISA was used to detect the levels of connective tissue growth factor （ CTGF ） and fibronectin （ FN） .Results： LF was induced into MF by TGF-β196 hours later.The immune fluorescence performance of α-SMA in the lung fibroblasts revealed MMF could suppress the expression of α-SMA,but had no effect on the phenotype of myofibroblasts .The results of ELISA showed that the levels of CTGF and FN were significantly decreased compar with that of control group and was concentration -de-pendent （ P〈0.05 ） .Conclusion： MMF can prevent lung fibroblasts from transdifferentiating into myofibroblasts and inhibit the expressions of CTGF and FN ,suggesting that MMF has anti-fibrosis effect and one of the mechanisms is by suppressing the expressions of CTGF and FN.