HGF对TGF-β1诱导尿道瘢痕成纤维细胞α-SMA及细胞外基质过度合成的保护作用

目的 探讨HGF对TGF-β1诱导尿道瘢痕成纤维细胞α-SMA及细胞外基质过度合成的保护作用.方法 收集尿道下裂术后瘢痕组织的标本,进行尿道瘢痕成纤维细胞的分离和培养.待细胞生长成单层后,以胰蛋白酶消化传代.取第四代成纤维细胞用于实验,当细胞达到80%融合时,培养液中加入TGF-β1(5ng/ml)及HGF(10～40ng/ml).培养72 h后,用RT-PCR检测各组α-SMA mRNA的变化;ELISA测定细胞Ⅰ、Ⅲ型胶原及纤维结合素的表达.结果 TGF-β1能显著诱导α-SMA m-RNA表达.随HGF的加入,α-SMA m-RNA的表达则明显受到抑制(P＜0.05),且随HGF浓度的升高其阻抑作用呈逐渐增强趋势.对照组、单纯TGF-β1组、加入TGF-β1与10、20、40ng/ml HGF组的Ⅰ型胶原A值分别为0.51±0.04、0.78±0.05、0.71±0.02、0.63±0.03、0.57±0.02,Ⅲ型胶原A值分别为0.12±0.01、0.29±0.02、0.21±0.02、0.14±0.01、0.08±0.01,纤维结合素A值分别为0.24±0.03、0.51±0.02、0.49±0.01、0.38±0.02、0.28土0.01.表明TGF-β1同样能诱导Ⅰ、Ⅲ型胶原及纤维结合素的表达(P＜0.01),而HGF则可以有效地阻抑其表达,其效应呈剂量依赖性(P＜0.05).结论 HGF对TGF-β1诱导的尿道瘢痕成纤维细胞α-SMA及细胞外基质过度合成具有抑制作用.这为临床预防和治疗尿道瘢痕狭窄提供了理论依据.

Abstract：

Objective To examine the inhibitory effects of HGF on TGF-β1 induced a-SMA and extracellular matrix synthesis in cultured fibroblasts derived from urethral scar. Methods Fibroblasts isolated from urethral scar were cultured ex vivo. After the fibroblasts reached confluence, cells were detached using trypsin/ethylenediamine tetra-acetic acid. All experiments were performed using the cells at the fourth passage. At 80% confluence, TGF-β1 (5 ng/ml) and HGF (10-40 ng/ml) were added to the culture medium. After 72 hours co-incubation, the mRNA of a-SMA was studied by RTPCR. The productions of collagen Ⅰ, Ⅲ and fibronectin in supernatants were also examined using ELISA. Results TGF-β1 markedly induced a-SMA mRNA expression in cultured fibroblasts. However, HGF could abrogated TGF-β1-induced a-SMA mRNA expression in a dose-dependent manner (P＜0. 05). The levels of collagen type Ⅰ were 0. 51 ± 0. 04,0. 78 ± 0. 05,0. 71 ± 0. 02,0. 63 ± 0. 03, and 0. 57 ± 0. 02 in control, TGF-β1, TGF-β1 + 10 ng/ml HGF, TGF-β1 + 20 ng/ml HGF, and TGF-β1 +10 ng/ml HGF group, respectively. The levels of collagen type Ⅲ were 0. 12 ± 0. 01,0. 29 ± 0. 02,0. 21 ± 0. 02,0. 14 ± 0. 01, and 0. 08 ± 0. 01 in control, TGF-β1, TGF-β1 + 10 ng/ml HGF, TGF-β1 +20 ng/ml HGF, and TGF-β1 + 10 ng/ml HGF group, respectively. And the levels of fibronectin were 0.24±0.03,0.51 ± 0.02,0.49 ± 0.01,0.38 ± 0.02,0.28 ± 0.01 in control, TGF-β1, TGF-β1 +10 ng/ml HGF, TGF-β1 + 20 ng/ml HGF, and TGF-β1 + 10 ng/ml HGF group, respectively. TGF-β1 significantly stimulated collagen Ⅰ, Ⅲ and fibronectin production in fibroblasts (P＜0. 01 ). However,HGF could reduced abrogated the up-regulation of collagen Ⅰ, Ⅲ and fibronectin induced by TGF-β1 in a dose-dependent manner (P＜0. 05). Conclusions HGF can effectively inhibit TGF-β1 induced aSMA and extracellular matrix synthesis in cultured fibroblasts derived from urethral scar.