乙肝病毒X蛋白通过上调NOX4的表达活化肝星状细胞

HBx Protein Activated Hepatic Stellate Cells Through Up-regulation of NOX4 Expression

目的:探讨乙肝病毒X蛋白(HBx)对肝星状细胞(HSC)活化的影响及其可能的分子机制。方法:构建稳定转染HBx基因的L0_2肝细胞(L0_2-HBx),Western Blot鉴定细胞株中HBx蛋白的稳定表达。以L0_2-HBx、转染空质粒(L0_2-pcDNA3.1)和未转染肝细胞(L0_2)的条件培养基,分别孵育肝星状细胞株LX-2,即LX-2/L0_2-HBx、LX-2/L0_2-pcDNA3.1、LX-2/L0_2,Western Blot检测上述各组细胞α-SMA (肝星状细胞活化标志)和NOX4的蛋白表达。以L0_2-HBx的条件培养基孵育LX-2细胞,分为转染si-NOX4(LX-2/L0_2-HBx+si-NOX4)、无关干扰片段(LX-2/L0_2-HBx+snc-RNA)、未转染(LX-2/L0_2-HBx)3组,Western Blot检测各组LX-2细胞的α-SMA蛋白表达。结果:L0_2-HBx中稳定表达HBx蛋白;LX-2/L0_2-HBx细胞中α-SMA和NOX4的蛋白表达显著高于LX-2/L0_2-pcDNA3.1、LX-2/L0_2细胞(P<0.01);LX-2/L0_2-HBx细胞敲减NOX4后(LX-2/L0_2-HBx+si-NOX4),α-SMA的蛋白表达显著低于LX-2/L0_2-HBx+snc-RNA和LX-2/L0_2-HBx细胞(P<0.05)。结论:肝细胞中表达的HBx蛋白可以通过HSC中NOX4上调α-SMA的表达,促进HSC的活化。

Objective: To explore the mechanism of HSC activation by HBxprotein. Methods: HBx-transfected human non-tumour hepatic L02 cell line, named L02-HBx was established and showed HBx expression by western blot analysis. Western blot tested the effects of HBx on NOX4 and α-SMA expression in human HSC HSC cell line(LX-2) after exposure to conditioned medium from L02-HBx, L02-pcDNA3.1, and L02 cells. After LX-2/L02-HBx cells treated with si-NOX4, the NOX4 and α-SMAexpression were tested by western blot analysis. Results: The L02-HBx cell models were successfully constructed. The expression of HBx was significantly expressed. Compared with LX-2/L02-pcDNA3.1 and LX-2/L0 groups, NOX4 and α-SMAexpression were increased significantly in LX-2/L02-HBx group(P<0.01). α-SMA in LX-2/L02-HBx expressions were depressed, coupled with the fact that the NOX4 was silenced(P<0.05). Conclusion: HBx protein activate hepatic stellate cells, and NOX4 play a vital role in the activation.