Inhibition of hepatocyte growth factor on proliferation and transdifferentiation of human Tenon capsule fibroblasts triggered by transforming growth factor-β1 in vitro / 中华实验眼科杂志

ABSTRACT

Objective To investigate the effects of hepatocyte growth factor (HGF) on proliferation and transdifferentiation of human Tenon capsule fibroblasts induced by transforming growth factor-β1(TGF-β1) in vitro.Methods Human Tenon capsule fibroblasts were cultured and divided into blank control group,TGF-β1 treated group and different concentrations HGF+TGF-β1 groups.The TGF-β1 at 10 μg/L was added into culture medium of the TGF-β1 treated group,and different concentrations of HGF (25,50,100,200 μg/L) and 10 μg/L TGF-β1 were added into culture medium of the HGF25 μg/L +TGF-β1,HGF50 μg/L +TGF-β1,HGF100 μg/L +TGF-β1,HGF200 μg/L +TGF-β1 group respectively,Methyl thiazolyl tetrazolium (MTT) was employed to measure the cell proliferation (absorbance at 560 nm).Immunofluorescence staining was used to evaluate and locate the expression of α-smooth muscle action (α-SMA) in the cells.The expression of α-SMA protein in the cells was detected by Western blot assay.Results Cultured cells showed fusiform in shape with the positive response for vimentin.The proliferation value of the cells was 0.203±0.025,0.497 ± 0.101,0.426 ± 0.062,0.354 ± 0.040,0.272 ± 0.084,0.241 ± 0.011 in the blank control group,TGF-β1 treated group,HGF25 μg/L +TGF-β1 group,HGF50 μg/L +TGF-β1 group,HGF100 μg/L +TGF-β group and HGF200 μg/L + TGF-β1 group,respectively,showing a significant difference among the groups (F =9.21 0,P =0.003).Compared with the TGF-β1 treated group,the proliferation values of the cells were significantly reduced in the blank control group and HGF+TGF-β1 group (all at P＜0.05).Immunofiuorescence staining showed that α-SMA protein mainly located in cytoplasm with the strong red fluorescence in the cells of the TGF-β1 treated group and weak red fluorescence in HGF+TGF-β1 group,and the expression of α-SMA was absent in the blank control group.The percentage of α-SMA-positive cells was (60.0±4.7)％ in the TGF-β1 treated group and (14.3±3.1)％ in the HGF+TGF-β1 group,with significant difference between the two groups (t =19.856,P＜0.001).The relative expression levels of the α-SMA protein in the cells were 0.642 ±0.032,1.330± 0.069 and 0.884 ±0.040 in the blank control group,TGF-β1 group and HGF100μg/L +TGF-β1 group,respectively,showing a significant difference among the groups (F =13.370,P＜ 0.001),and relative expression levels of the α-SMA protein in the cells were significantly lower in the blank controlgroup and HGF100 μg/L+TGF-β1 group than that in the TGF-β1 treated group (all at P＜0.05).Conclusions HGF can inhibit the proliferation of human Tenon capsule fibroblasts,down-regulate the expression of α-SMA protein induced by TGF-β1 and arrest the phenotype transformation of fibroblasts in vitro.