血红素加氧酶-1对肝癌细胞细胞周期调控因子的影响

目的 观察血红素加氧酶-1(heme oxygenase 1,HO-1)对人肝癌细胞HepG2细胞周期调控因子的影响.方法 构建含有野生型和突变型HO-1基因的重组载体pcDNA3.1(+)-wtHO-1和pcDNA3.1(+)-mHO-1G143H.利用脂质体介导的方法将构建好的重组载体转染肝癌细胞系HepG2,以空载体转染作为对照组.通过G418筛选建立稳定表达野生型和突变型HO-1的HepG2肝癌细胞系.经半定量RT-PCR、Western印迹检测转染细胞系中HO-1 mRNA和蛋白的表达水平.在HO-1表达改变的稳转细胞系中,利用Western 印迹检测转染细胞系中P21、P27蛋白表达水平.结果 成功实现了野生型和突变型HO-1在HepG2细胞中的过表达;野生型和突变型HO-1过表达均能诱导抑癌基因p21和p27的表达.结论 HO-1过表达诱导抑癌基因p21和p27的表达与血红素分解产物无关.HO-1可能通过其它机制调节p21和p27的表达.

Effect of Heme Oxygenase-1 on Cell Cycle Regulatory Factors in Liver Cancer Cells

Objective To elucidate the effect of heme oxygenase-1 on cell cycle regulator in HepG2 cell line.Methods To construct eukaryotic expression vectors expressing wild-type HO-1 and G143H mutant HO-1,named pcDNA3.1 (+) -wtHO-1 and pcDNA3.1 (+) - mHO-1G143H. HepG2 cells were transfected with wtHO-1 and mHO-1 using lipofectamine 2000. Stable transfected cells were selected by G418. Expression of HO-1 mRNA and protein were detected using RT-PCR and Western blot respectively. In addition,expression of p21 and p27,the major cyclin-dependent kinase(Cdk)inhibitors in the two cell lines were examined.Results HepG2 cell lines with wild type and mutant HO-1 overexpression were established successfully. Overexpression of both wild type and mutant HO-1 enhanced the expression of tumor suppressor P21 and P27.Conclusion HO-1 overexpression upregulated the level of P21 and P27. This regulation is not dependent of HO-1 catalytic activity,but probably other mechanisms.