乙型肝炎病毒促进CTGF和TGF-β1在肝星状细胞中的表达

Hepatitis B virus induces expression of connective tissue growth factor and transforming growth factor-β1 in hepatic stellate cells

目的:研究转染乙型肝炎病毒(HBV)的HepG2.2.15细胞株在体外促进肝星状细胞中CTGF和TGF-β1的表达,进而探讨HBV促肝细胞纤维化的机制.方法:将HepG2和HepG2.2.15细胞株分别在体外与肝星状细胞(LX-2)共培养,以单独培养的肝星状细胞为对照组.培养24,48,72 h后,以Real-time PCR定量检测肝星状细胞中CTGF和TGF-β1 mRNA的表达,以Western blot定量检测其蛋白表达结果:与对照组比较,在24、48、72 h时点,CTGF和TGF-β1 mRNA分别增高约1.7、4.2、9.6倍(P<0.01)和2.2、6.1、8.1倍(P<0.01),以72 h差异最为显著;而与HepG2细胞共培养实验组LX-2细胞CTGF和TGF-β1 mRNA在三个时间点分别增高约1.7、1.2、1.3倍(P<0.05)和2.7、1.9、2.1倍(P<0.05).CTGF和TGF-β1蛋白表达量分别增高约2.1、2.6、2.5倍(P<0.01)和1.7、3.3、3.1倍(P<0.01),以48 h差异最为显著:而与HepG2细胞共培养实验组LX-2细胞CTGF和TGF-β1蛋白表达量在三个时间点分别增高约1.6、1.1、0.9倍(P<0.05)和1.1、1.4、2.5倍(P<0.05).结论:与HepG2.2.15细胞株共培养后,肝星状细胞中肝纤维化相关因子的表达明显增强.体外实验证明HBV具有诱导肝细胞纤维化的重要作用.

AIM： To investigate whether HepG2.2.15 cell line induces the expression of connective tissue growth factor （CTGF） and transforming growth factor-β1 （TGF-β1） in hepatic stellate cells and the mechanism of hepatitis B virus （HBV） in inducing fibrogenesis. METHODS： The hepatic stellate cells （LX-2） were co-cultured with HepG2 or HepG2.2.15 in vitro and the LX-2 cells cultured alone were used as controls. After culturing for 24, 48 and 72 h, real-time polymerase chain reaction （PCR） was performed to detect the expression of CTGF and TGF-β1 mRNA in LX-2 cells. Western-blot analysis was used to measure the expression of CTGF and TGF-β1 proteins in LX-2 cells. RESULTS： After 24, 48, and 72 h, the expression of CTGF and TGF-β1 mRNA in LX-2 cells co-cultured with HepG2.2.15 were higher than those in the controls （CTGF： 1.7, 4.2, 9.6 times higher, P 〈 0.01; TGF-β1： 2.2, 6.1, 8.1 times higher, P 〈 0.01）, and the most eminent effect was found at 72 h; however, CTGF and TGF-β1 mRNA expression in LX-2 cells co-cultured with HepG2 were 1.7, 1.2, 1.3 and 2.7, 1.9, 2.1 times higher than those in the controls （all P 〈 0.05）. At the same time point, the protein expression of CTGF and TGF-β1 in LX-2 cells co-cultured with HepG2.2.15 （CTGF： 2.1, 2.6, 2.5 times higher, P 〈 0.01; TGF-β1： 1.7, 3.3, 3.1 times higher, P 〈 0.01） or HepG2 （CTGF： 1.6, 1.1, 0.9 times higher, P 〈 0.05; TGF-β1： 1.1, 1.4, 2.5 times higher, P 〈 0.05） were also higher than those in the control cells. CONCLUSION： The expression of fibrosis-related factors in hepatic steUate cells are increased significantly after co-culturing with HepG2.2.15, which proves that HBV can induce fibrogenesis in vitro.