目的 为研究线粒体融合基因2(mitofusin-2,Mfn2)的异常表达对人胶质瘤的影响,构建Mfn2特异性表达的RNAi慢病毒载体,并以此构建Mfn2沉默的稳定人胶质瘤细胞株.方法 设计Mfn2 mRNA的特异性siRNA,构建RNAi慢病毒载体质粒并进行测序鉴定.利用构建好的RNAi慢病毒载体质粒以及pHelper 1.0载体和pHelper 2.0载体3种质粒共转染293T细胞,包装慢病毒并测定滴度.用包装好的慢病毒感染人脑胶质瘤U87MG细胞株,利用RT-PCR及Western blot测定细胞中Mfn2的转录及翻译情况;利用CCK-8实验及平板克隆形成实验检测Mfn2-RNAi对U87MG胶质瘤细胞增殖的影响.结果 质粒测序结果与目的序列一致,提示RNAi序列正确插入Mfn2基因;慢病毒包装成功,病毒滴度为4×108 TU·mL-1;病毒感染后U87MG细胞生长状态良好,荧光持续稳定表达,Mfn2转录及翻译受到抑制,LV-Mfn2-RNAi组较对照组细胞增多(P<0.05).结论 人Mfn2基因RNAi慢病毒构建成功;Mfn2沉默的U87MG细胞株构建成功;Mfn2表达降低可能促进胶质瘤细胞增殖.%Objective To investigate the effect of abnormal expression of Mfn2 gene on human glioma and to construct the lentiviral vector harboring RNA interference sequence targeting mitofusin-2(Mfn2)and with whom to construct the Mfn2 stably silent human glioma cell line.Methods The specific siRNA of Mfn2 mRNA was designed,and the plasmids of RNAi lentiviral vector was constructed and identified by DNA sequencing. The 293T cells were co-transfected with the plasmids of RNAi lentiviral vector,pHelper1.0 and pHelper2.0 in order to produce virus and determine the titer. The RT-PCR and Western Blotting were used to measure the expression of Mfn2 in human glioma U87MG cell line which were infected by the virus. The CCK-8 assay and plate cloning formation method were used to test the proliferation of U87MG cell line which were infected by the virus.Results DNA sequencing that the DNA sequences of the plasmids were consisted with the design suggested that the RNAi sequence was inserted correctly into the Mfn2 gene. The lentiviral were produced successfully with the titer of 4×108TU·mL-1. The U87MG cells grew well with strong green fluorescence after infection and the Mfn2 was significantly inhibited(P<0.05),which suggested that the U87MG cell line with Mfn2 silencing was successfully constructed. The increased cell number of the LV-Mfn2-RNAi group was significantly more than those of the other two control groups.Conclusion The RNAi lentiviral vector of the Mfn2 gene of Homo sapiens was successfully constructed and the inhibition of Mfn2 could promote the proliferation of glioma cell.
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