摘要目的 研究肝细胞生长因子(HGF)对转化生长因子-β1(TGF-β1)诱导的人Tenon囊成纤维细胞增生和转分化的影响.方法 人Tenon囊成纤维细胞进行常规培养后分为空白对照组、TGF-β1处理组及不同质量浓度HGF+TGF-β1组.TGF-β1处理组在细胞培养液中添加10 μg/L TGF-β1,不同质量浓度HGF+TGF-β1组在细胞培养液中分别添加10 μg/L TGF-β1,然后分别添加不同质量浓度的HGF(25、50、100、200 μg/L),采用甲基偶氮四唑(MTT)法检测波长560 nm处各组细胞的吸光度(A560);然后选用100 μg/L的HGF进行干预,采用细胞免疫荧光染色技术检测人Tenon囊成纤维细胞中0-平滑肌肌动蛋白(α-SMA)的表达分布;采用Western blot法检测细胞中α-SMA蛋白的相对表达量. 结果 体外培养的人Tenon囊成纤维细胞呈长梭形,边界清楚,细胞中波形蛋白表达阳性并定位于细胞质.MTT检测显示空白对照组、TGF-β1处理组及HGF25μg/L +TGF-β1组、HGF50μg/L+TGF-β1组、HGF100μg/L+TGF-β1组、HGF200μg/L+TGF-β1组细胞的增生值分别为0.203±0.025、0.497±0.101、0.426±0.062、0.354±0.040、0.272±0.084和0.241±0.011,组间比较差异有统计学意义(F=9.210,P=0.003),TGF-β1处理组细胞增生值明显高于空白对照组,不同质量浓度HGF+TGF-β1组细胞增生值均明显低于TGF-β1处理组,差异均有统计学意义(均P＜0.05).免疫荧光染色结果显示空白对照组细胞中未见α-SMA的表达,TGF-β1处理组及HGF100μg/L+TGF-β1组细胞的胞质中均可见α-SMA的表达,呈红色荧光,HGF100μg/L+TGF-β1组细胞中α-SMA表达荧光减弱,α-SMA表达细胞明显减少.TGF-β1处理组和HGF100μg/L+TGF-β1组细胞中α-SMA染色阳性细胞百分比分别为(60.0±4.7)％和(14.3±3.1)％,差异有统计学意义(t=19.856,P＜0.001).Western blot检测显示空白对照组、TGF-β1处理组和HGF100μg/L+TGF-β1组细胞中α-SMA蛋白相对表达量分别为0.642±0.032、1.330±0.069和0.884±0.040,总体比较差异有统计学意义(F=13.370,P＜0.001),其中TGF-β1处理组细胞中α-SMA蛋白相对表达量明显高于空白对照组,HGF100μg/L+TGF-β1组细胞中α-SMA蛋白相对表达量明显低于TGF-β1处理组,差异均有统计学意义(均P＜0.05). 结论 HGF抑制TGF-β1诱导的人Tenon囊成纤维细胞的过度增生,下调细胞中α-SMA蛋白的表达,阻止成纤维细胞的表型转化.

abstractsObjective 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,HGFs0 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,HGFs0 μg/L +TGF-β1 group,HGF100 μg/L +TGF-β1 group 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).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.