摘要
目的:研究外源性线粒体融合素基因-2(mitofusin-2gene,mfn2)对肝癌细胞株HepG2体外生物学行为的影响。方法:基因重组构建并鉴定mfn2真核表达质粒pEGFPmfn2,用脂质体将质粒转染培养人肝癌细胞株HepG2,经G418筛选阳性细胞克隆,逆转录-聚合酶链反应(RT-PCR)检测转染后细胞mfn2mRNA的表达水平,Western-blot检测线粒体融合蛋白的表达,四甲基偶氮唑蓝(MTT)法检测mfn2对HepG2细胞增殖的影响,Annexin-V/PI双标流式细胞术检测转染细胞凋亡的变化。结果:重组真核表达质粒pEGFPmfn2经限制性内切酶双酶切,电泳后显示约2300bp的mfn2片段和4700bp的pEGFP-N2载体片段。RT-PCR及Western-blot显示转染pEGFPmfn2组有mfn2基因mRNA及其蛋白表达。MTT实验提示转染mfn2基因后,HepG2细胞增殖受到抑制;流式细胞分析显示转染pEGFPmfn2组与转染pEGFP组和空白对照组相比可促进细胞凋亡。结论:成功构建了pEGFPmfn2真核表达质粒,外源性mfn2基因可抑制肝癌细胞的增殖,并可诱导肝癌细胞凋亡。
Objective To investigate the effects of mitofusin-2 gene （mfn2） on the biological behaviors of hepati carcinoma cell line HepG2 in vitro. Methods The recombinant eukaryotic expressing plasmidc pEGFPmfn2 was constructed, identified by using recombinant DNA technique, and then transfected into HepG2 cells. After the transfected cells were screened with G418, the mRNA expression of the cell was detected by RT-PCR, so was the protein expression by Western-blot. The proliferation of HepG2 was measured by MTF and apoptosis was assessed by Annexin-V/PI double staining flow cytometry. Results The plasmid pEGFPmfn2 was digested by restriction enzyme and eleetrophoresis of the products showed a mfn2 fragment of 2 300 bp and a pEGFP-N2 of 4 700 bp. The expressions of mfn2 mRNA and protein were detected by RT-PCR and Western-blot. Cell proliferation was inhibited following mfn2 transfection. Flow cytometry assay indicated that the apoptotic rate was increased in the transfected group, as compared with other groups. Conclusions The recombinant eukaryotic expressing plasmid pEGFPmfn2 has been successfully constructed. Exogenous mfn2 gene can inhibit the proliferation of hepatic carcinoma cell and induce apoptosis of the cell.
出处
《实用医学杂志》
CAS
2008年第20期3462-3465,共4页
The Journal of Practical Medicine
关键词
癌细胞胞
线粒体基因
基因转染
细胞凋亡
Carcinoma,hepatocellular Mitochondrial gene Gene transfeetion Apoptosis
