Authors: Chen Jing, Wang Yong, Li Donghao
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.
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.
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-β1group and HGF200 μg/L+ TGF-β1 group, respectively, showing a significant difference among the groups (F=9.210, 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). Immunofluorescence 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 HGF100 μg/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 control group and HGF100 μg/L+ TGF-β1 group than that in the TGF-β1 treated group (all at P<0.05).
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.