线粒体内膜蛋白质mitofilin干涉慢病毒的构建及应用

Construction of a lentiviral vector for mitochondria inner membrane protein mitofilin gene silencing and its application

目的构建线粒体内膜蛋白质mitofilin的慢病毒干涉载体,初步研究mitofilin在体内体外的生物学功能。方法将靶向人mitofilin基因的短发夹RNA(shRNA)序列克隆到慢病毒核心质粒pSicoR中,瞬时转染HEK293T细胞筛选出有效的干涉片段,并进行慢病毒的包装。病毒感染人宫颈癌细胞系HeLa细胞,Western印迹检测mitofilin蛋白的干涉效果。15 Gyγ射线照射刺激病毒感染的HeLa细胞,流式细胞术检测细胞凋亡。利用尾静脉大容量快速注射法将慢病毒注射入C57BL/6J小鼠体内,6.5 Gyγ射线照射刺激,蓝色温和电泳联合胶内酶活性染色分析,检测呼吸链复合体Ⅴ活性变化。结果与结论成功构建具有mitofilin干涉效果的慢病毒干涉载体并获得了稳定干涉mitofilin的HeLa细胞株。构建的慢病毒能有效下调HeLa细胞和小鼠肝脏细胞mitofilin蛋白的表达。干涉mitofilin表达可增加γ射线诱导的细胞凋亡,降低γ射线刺激下小鼠肝细胞线粒体呼吸链复合体Ⅴ的活性。所构建的干涉慢病毒能够广泛应用于mitofilin在体内和体外的功能研究。

Objective To construct a lentiviral vector for interfering the expression of human mitofilin gene,and explore the biological function of mitofilin both in vivo and in vitro.Methods Four different short-hairpin RNA（shRNA） s targeting human mitofilin gene were cloned into pSicoR vectors to obtain the recombinant plasmid.The recombinant vectors were transfected into HEK293T cells and the effective silencing vector for lentiviral packaging was chosen.The HeLa cells were infected with lentivirus and the inhibiting effects of mitofilin were detected by Western blot.The apoptosis rate of HeLa cells infected with lentivirus after treated with 15 Gy γ-irradiation was detected.The lentivirus was injected into C57BL / 6J mice by high volume tail-vein injection.The mitochondrial protein was extracted and the changes in respiratory chain complex Ⅴ activity was measured by BN-PAGE combined with in-gel catalytic activity assays.Results and Conclusion A lentiviral vector carrying an shRNA targeting the mitofilin gene was successfully constructed and the HeLa cell line stably inhibiting mitofilin expression was established.Western blot analyses confirmed that the lentivirus constructed could effectively inhibit mitofilin expression in both HeLa cells and mice liver.Further study showed that,down-regulated mitofilin could increase the cell apoptosis rate induced by γ-irradiation and the activity of ATP synthase in mouse mitochondria under γ-irradiation was remarkably reduced.The lentiviral system for mitofilin gene silencing could be widely used in the functional study of mitofilin both in vivo and in vitro.