线粒体内膜蛋白质mitofilin干涉慢病毒的构建及应用

发布时间：2018-01-03 01:32

  本文关键词：线粒体内膜蛋白质mitofilin干涉慢病毒的构建及应用　出处：《军事医学》2013年09期 　论文类型：期刊论文

  更多相关文章： 线粒体内膜蛋白 慢病毒 凋亡 线粒体 ATP合酶

【摘要】：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。
[Abstract]:Objective to construct the lentivirus interference vector of mitochondrial intima protein mitofilin. To study the biological function of mitofilin in vitro and in vivo. Methods the short hairpin RNAs targeting the human mitofilin gene were used. The sequence was cloned into the core plasmid pSicoR of lentivirus. Transient transfection of HEK293T cells to screen effective interference fragments and packaging of lentivirus. The virus infected human cervical cancer cell line HeLa cells. Western blot was used to detect the interference effect of mitofilin protein. 15 Gy 纬 -ray irradiation was used to stimulate HeLa cells infected by virus. Apoptosis was detected by flow cytometry. Lentivirus was injected into C57BL / 6J mice by rapid mass injection of caudal vein. Blue gel electrophoresis combined with gel enzyme activity staining analysis. Results and conclusion Lentivirus interference vector with mitofilin interference effect was successfully constructed and HeLa cells with stable interference mitofilin were obtained. The constructed lentivirus could effectively down-regulate the expression of mitofilin protein in HeLa cells and mouse liver cells. Interfering with the expression of mitofilin could increase the apoptosis induced by 纬 -rays. It can reduce the activity of mitochondrial respiratory chain complex 鈪，

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