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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 mitochondrial lentiviral vector of mitofilin, and to study the biological function of mitofilin in vitro and in vivo. Methods The short hairpin RNA (shRNA) sequence targeting human mitofilin gene was cloned into the lentiviral core plasmid pSicoR. The recombinant plasmid was transiently transfected into HEK293T cells for screening of effective interfering fragments and for lentivirus packaging. Human cervical cancer cell line HeLa was infected with virus and the interference effect of mitofilin protein was detected by Western blotting. HeLa cells infected by virus were stimulated by 15 Gy γ-rays, and apoptosis was detected by flow cytometry. The lentivirus was injected into C57BL / 6J mice by rapid injection of tail vein, and the changes of V activity of respiratory chain complex were detected by 6.5Gy radiation, blue mild electrophoresis and gel electrophoresis staining. RESULTS AND CONCLUSION: Lentiviral interference vectors with mitofilin interference were successfully constructed and HeLa cell lines stably interfering with mitofilin were obtained. The constructed lentivirus can effectively down-regulate mitofilin expression in HeLa cells and mouse liver cells. Interfering mitofilin expression increased γ-ray-induced apoptosis and decreased the activity of mitochondrial respiratory chain complex Ⅴ in mouse hepatocytes stimulated by γ-rays. The constructed lentivirus can be widely used to study the function of mitofilin in vivo and in vitro.