LMP-1基因慢病毒载体构建及其在大鼠骨髓间充质干细胞的表达  

Construction of lentivirus vector containing human LIM mineralization protein-1(LMP-1)and its expression in rat bone mesenchymal stem cells

作　　者：侯慧铭[1] 向川[1] 郭丽[1] 张桦栋[1] 

机构地区：[1]山西医科大学第二医院骨科,山西太原030001

出　　处：《中国骨伤》2013年第10期841-844,共4页China Journal of Orthopaedics and Traumatology

基　　金：山西省自然科学基金资助项目(编号:2010011050-2)~~

摘　　要：目的:构建人LMP-1重组慢病毒载体,体外转染大鼠骨髓间充质干细胞,检测LMP-1基因在大鼠骨髓间充质干细胞的表达。方法:利用PCR法从cDNA文库中钓取LMP-1基因,将其与经AgeⅠ酶切线性化的慢病毒载体pGC-FU-EGFP相连接,转化感受态大肠杆菌,筛选出阳性克隆pGC-FU-LMP-1-EGFP,基因测序对其鉴定。经293T细胞包装后,收集富含病毒颗粒LV-LMP-1-EGFP的细胞上清,浓缩并标定滴度,RT-PCR检测并鉴定。以最佳MOI值体外转染大鼠骨髓间充质干细胞,荧光显微镜观察转染是否成功,流式细胞仪检测转染效率,RT-PCR和Western blot检测转染细胞LMP-1基因的表达。结果:①基因测序及RT-PCR检测证实携带人LMP-1基因的慢病毒载体构建成功,包装后获得滴度为2×108TU/ml的LV-LMP-1-EGFP。②以MOI=100转染大鼠骨髓间充质干细胞,荧光显微镜下可见大量绿色荧光蛋白表达,转染效率可达93.5%,经RT-PCR与Western blot检测,被转染细胞内有LMP-1基因表达。结论:成功构建携带人LMP-1基因的慢病毒载体,可高效转染大鼠骨髓间充质干细胞,被转染细胞可高效表达LMP-1基因。Objective ：To construct a recombiant lentivirus vector of human LMP-1 and detect the expression of LMP-1 in infected rat bone mesenchymal stem cells. Methods ：LMP-1 gene from the cDNA library were extracted by Polymerase Chain Reaction （PCR）. The LMP-1 genes were connected into lentiviral vectors pGC-FU-EGFP which was linearized by Age I enzyme to produce recombiant ｜entivirus vector called as pGC-FU-LMP-1-EGFP ,then packaged by 293T cells. The virus supernant congtaining LV-LMP-1-EGFP was harvested,concentrated and titrated. The rat BMSCs were transfected with re- combiant lentivirus LV-LMP-1-EGFP at the most appropriate MOI. The mRNA and protein expression of LMP-1 were detected by RT-PCR and Western blot. Results： ①LV-LMP-1-EGFP was recombined successfully and the titer reached 2×10^8 TU/ml. ②The efficiency of infection was 93.5% ,which was get after LV-LMP-1-EGFP infected rat BMSCs at the most appropriate MOI=100. The expression of LMP-1 gene was confirmed by RT-PCR and Western blot. Conclusion：Lentivirus vector con- taining human LMP-1 gene is constructed successfully,which can transfected efficiently into rat BMSCs, and the infected rat BMSCs can effectively express LMP-1.

关 键 词：慢病毒 LMP-1 骨髓间充质干细胞 骨质疏松 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]