线粒体融合素基因2联合氟尿嘧啶对肝癌细胞的作用

The effects of combination of fluorouracil with mitofusin-2 gene transfection on hepatocellular carcinoma cells

目的研究线粒体融合素基因2(mfn2)转染联合氟尿嘧啶对肝癌细胞的作用。方法用脂质体2000将质粒pEGFPmfn2、空质粒pEGFP N2分别转染HepG2细胞,实验分三组:重组质粒pEGFPmfn2转染HepG2细胞为实验组,空质粒pEGFP-N2转染HepG2细胞为阴性对照组,HepG2细胞为空白对照组。RT-PCR和Western Blot分别检测转染后各组细胞mRNA和蛋白水平的表达。采用MTT法检测mfn2基因转染联合5-氟尿嘧啶对肝癌细胞增殖的影响;流式细胞术检测转染后细胞周期的分布情况。结果经脂质体2000转染后,RT-PCR结果显示转染mfn2基因的HepG2细胞基因组中存在有目的基因特异性片段,表明转染成功;Western Blot检测结果表明转染后细胞中有Mfn2蛋白表达;流式细胞术对细胞周期分布检测的结果表明,细胞被阻滞在G0/G1期,转染pEGFPmfn2组G0/G1期所占比例为(83.2±1.5)%,明显高于转染空质粒pEGFP组与对照组G0/G1期所占比例(P<0.05)。MTT法检测结果提示转染mfn2基因的HepG2细胞可增强化疗药物氟尿嘧啶对细胞的增殖抑制作用。结论线粒体融合素基因2联合氟尿嘧啶作用肝癌细胞能够加强氟尿嘧啶的增殖抑制作用,明显增强肝癌细胞对氟尿嘧啶的敏感性。

Objective To investigate the effect of combination of fluorouracil with mitofusin-2 gene transfection on hepatocellular carcinoma cells.Methods Plasmid pEGFPmfn2 and pEGFP-N2 were transfected respectively into hepatocellular carcinoma cell strain HepG2 by Lipofectamine TM 2000 in vitro.There were three groups: pEGFPmfn2 cells,pEGFP cells and HepG2 cells.The mRNA expression was detected by RT-PCR in cells and protein expression by Western-blot assay.MTT assay were used to detect the cell proliferation of combination of fluorouracil with mitofusin-2 gene transfection on hepatocellular carcinoma cells.Cell cycle of HepG2 cells was observed by flow cytometry.Results After transfected by Lipofectamine TM 2000,RT-PCR revealed that the genome of mfn2-transfected cells contained a 392bp specific fragment of mfn2 gene which indicated the successful transfection,and successful protein expression was confirmed by Western Blot assay.Flow cytometry assay showed that in the cell cycle the G0/G1 phase proportion was significant higher in pEGFPmfn2-transfected cells(83.2±1.5)% than in pEGFP-transfected cells and untransfected cells after transfection(P<0.05).The results indicated that exogenous mfn2 gene could increase their chemosensitivity to 5-Fu.Conclusion The combination of fluorouracil with mfn2 gene could increase the inhibitory function of fluorouracil and sensitize HepG2 cells to fluorouracil.