本文關(guān)鍵詞：線粒體內(nèi)膜蛋白質(zhì)mitofilin干涉慢病毒的構(gòu)建及應(yīng)用　出處：《軍事醫(yī)學(xué)》2013年09期 　論文類型：期刊論文

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【摘要】：目的構(gòu)建線粒體內(nèi)膜蛋白質(zhì)mitofilin的慢病毒干涉載體,初步研究mitofilin在體內(nèi)體外的生物學(xué)功能。方法將靶向人mitofilin基因的短發(fā)夾RNA(shRNA)序列克隆到慢病毒核心質(zhì)粒pSicoR中,瞬時(shí)轉(zhuǎn)染HEK293T細(xì)胞篩選出有效的干涉片段,并進(jìn)行慢病毒的包裝。病毒感染人宮頸癌細(xì)胞系HeLa細(xì)胞,Western印跡檢測mitofilin蛋白的干涉效果。15 Gyγ射線照射刺激病毒感染的HeLa細(xì)胞,流式細(xì)胞術(shù)檢測細(xì)胞凋亡。利用尾靜脈大容量快速注射法將慢病毒注射入C57BL/6J小鼠體內(nèi),6.5 Gyγ射線照射刺激,藍(lán)色溫和電泳聯(lián)合膠內(nèi)酶活性染色分析,檢測呼吸鏈復(fù)合體Ⅴ活性變化。結(jié)果與結(jié)論成功構(gòu)建具有mitofilin干涉效果的慢病毒干涉載體并獲得了穩(wěn)定干涉mitofilin的HeLa細(xì)胞株。構(gòu)建的慢病毒能有效下調(diào)HeLa細(xì)胞和小鼠肝臟細(xì)胞mitofilin蛋白的表達(dá)。干涉mitofilin表達(dá)可增加γ射線誘導(dǎo)的細(xì)胞凋亡,降低γ射線刺激下小鼠肝細(xì)胞線粒體呼吸鏈復(fù)合體Ⅴ的活性。所構(gòu)建的干涉慢病毒能夠廣泛應(yīng)用于mitofilin在體內(nèi)和體外的功能研究。
[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 緯 -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 緯 -rays. It can reduce the activity of mitochondrial respiratory chain complex 鈪，

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