逆转录病毒介导的反义c-myb对肝星状细胞增殖及Ⅰ型胶原基因表达的影响

目的:探讨反义c-mybRNA对体外培养的肝星状细胞(HSC)增殖及Ⅰ型胶原mRNA表达的影响。方法:构建含有反向c-myb基因片段的重组逆转录病毒载体pDOR-myb,将其导入包装细胞PA317中,收获含病毒的培养上清,进一步感染体外培养的大鼠HSC,采用四甲基偶氮唑盐(MTT)法测定细胞增殖反应,采用半定量RT-PCR检测c-myb、α1-Ⅰ型胶原mRNA表达。结果:成功分离培养大鼠HSC,感染pDOR-myb病毒的HSC自身c-myb表达、细胞增殖及α-Ⅰ型胶原mRNA表达显著受抑。结论:c-myb在HSC激活增殖过程中起重要作用,反义c-myb基因能抑制HSC增殖及Ⅰ型胶原基因表达,这提示抑制c-myb表达可能是防治肝纤维化的有效途径。

Effects of c-myb antisense RNA on the proliferation and collagen Ⅰ gene expression in cultured hepatic stellate cells

AIM:To investigate the effects of c-myb antisense RNA on the proriferation and collagen Ⅰ gene expression in cultured hepatic stellate cells(HSC) in rats.METHODS:The c-myb antisense gene recombinant retroviral vector(pDOR-myb) was constructed, and then was transfected into retroviral package cell line PA 317 by means of N-[1-(2,3-Dioleoyloxy) propyl]-N, N, N-trimethylammonium methyl-sulfate(DOTAP) liposomal transfection reagent. The pseudoviruses produced from the resistant PA317 cells selected with G418 were collected, with which HSCs isolated from rat liver were infected. The cell proliferation was measured by MTT method, c-myb, α₁-Ⅰ collagen mRNA expression and c-myb protein in HSCs were detected with semi-quantitative RT-PCR and western-blot, respectively.RESULTS:HSCs from rats were isolated successfully with the viability >98%. In the pDOR-myb infected HSCs, c-myb expression levels, the cell proliferation, and α₁-Ⅰ collagen mRNA expression were repressed significantly.CONCLUSIONS:c-myb plays a key role in the activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation and α₁-Ⅰ collagen mRNA expression in the infected HSC. These data suggest that inhibition of c-myb gene expression would be a potential way for the treatment of liver fibrosis.