肝细胞生长因子/转化生长因子-β1对人心房成纤维细胞增殖的作用

Effects of hepatocyte growth factor and transforming growth factor-β1 on proli-feration of primarily cultured human atrial fibroblasts

摘要:

目的 探讨肝细胞生长因子(HGF)和转化生长因子-β₁(TGF-β₁)在体外培养的人心房成纤维细胞增殖中的作用,观察HGF对人心房成纤维细胞TGF-β₁表达的影响。方法 入选福建省立医院心血管外科接受换瓣手术的风湿性心脏病(RHD)患者20例,其中窦性心律(SR)者和慢性心房颤动(CAF)者各10例,分别作为SR组和CAF组。记录患者左心房内径(LAD)。换瓣术中无菌条件下取适量右心耳组织,人心房成纤维细胞原代培养。水溶性磷酸化四唑盐-1(WST-1)比色法检测HGF和TGF-β₁在不同浓度(HGF:25,50,100,200 ng/ml；TGF-β₁:0.1,1,10,100 ng/ml)和不同时间(12,24,48,72 h)对人心房成纤维细胞增殖的影响。检测HGF/TGF-β₁对人心房成纤维细胞增殖的影响。半定量逆转录-聚合酶链反应(RT-PCR)技术检测心房成纤维细胞TGF-β₁ mRNA水平,分为3组:SR组、CAF组和HGF干预组(CAF组中再加入100 ng/ml HGF干预48 h)。采用SPSS 19.0软件进行数据处理。两组间比较采用t检验,多组间比较采用单因素方差分析。结果 CAF组患者的LAD显著高于SR组[(5.76±1.37) vs(4.43±0.77)cm]。HGF和TGF-β₁对人心房成纤维细胞的增殖分别具有抑制和促进作用,且与剂量(rHGF＝-0.686；rTGF-β₁＝0.655)和时间(rHGF＝0.557；rTGF-β₁＝0.840)显著相关(P＜0.05),其中:100 ng/ml HGF作用48 h抑制作用最显著；10 ng/ml TGF-β₁作用48 h促进作用最显著。HGF/TGF-β₁对人心房成纤维细胞增殖具有抑制作用。与CAF组相比(1.78±0.98),SR组(0.92±0.66)和HGF干预组(0.67±0.64)的TGF-β₁ mRNA表达水平均显著减少,差异具有统计学意义(P<0.01)。结论 CAF患者LAD增大,TGF-β₁分泌增多。HGF能够抑制人心房成纤维细胞TGF-β₁的生成,部分拮抗TGF-β₁促增殖作用。

Abstract:

Objective To investigate the role of hepatocyte growth factor (HGF) and transforming growth factor beta 1 (TGF-β₁) on the proliferation of human atrial fibroblasts cultured in vitro, and determine the effect of HGF on the expression of TGF-β₁ in human atrial fibroblasts. Methods A total of 20 patients with rheumatic heart disease (RHD) who underwent cardiac valve replacement in the Department of Cardiovascular Surgery of Fujian Provincial Hospital were recruited in this study. They were 10 cases of sinus rhythm (SR) and 10 cases of chronic atrial fibrillation (CAF), and were assigned into as SR group and CAF group accordingly. The data of left atrial diameter (LAD) were collected for each patient. After moderate right atrial appendage tissues were obtained during the operation, the atrial fibroblasts were isolated and primarily cultured. Then the obtained cells were treated with different concentrations of HGF (25,0, 100 and 200 ng/ml) and TGF-β₁ (0.1,1, 10 and 100 ng/ml) for different times (12,4, 48 and 72 h), the effects of the concentrations and time periods on the proliferation of atrial fibroblasts were studied by water-soluble sulfonated tetrazolium (WST-1). The expression of TGF-β₁ at mRNA level in atrial fibroblasts from the SR, CAF and HGF intervention group (the cells from CAF group after 100 ng/ml HGF treatment for 48 h) were analyzed with RT-PCR. SPSS statistics 19.0 was used for data processing. Student’s t test was used for the comparison between groups, and single factor analysis of variance was used for intergroup comparison. Results LAD was significantly larger in the CAF patients than the SR patients [(5.76±1.37) vs (4.43±0.77)cm]. HGF and TGF-β₁ respectively inhibited and promoted the proliferation of human atrial fibroblasts in dose-dependent (rHGF=-0.686, rTGF-β₁=0.655,P<0.05) and time-dependent manners (rHGF=0.557, rTGF-β₁=0.840, P<0.05), with a peak action of 100 ng/ml HGF for 48 h, and of 10 ng/ml TGF-β₁ for 48 h. TGF-β₁ plus HGF showed inhibitory effect on cell proliferation. The mRNA expression level of TGF-β₁ was decreased in both SR group (0.92±0.66) and HGF intervention group (0.67±0.64) when compared with CAF group (1.78±0.98, P<0.01). Conclusion The CAF patients have larger LAD and increased TGF-β₁ expression. HGF inhibits TGF-β₁ expression in human atrial fibroblasts, and partially counteracts the promoting role of TGF-β₁ on cell proliferation.