慢病毒转导增强型绿色荧光蛋白对大鼠骨髓间充质干细胞生物学特性的影响  

Characteristics of rat BMSCs transfected with eGFP by using lentiviral vectors

作　　者：蒋涛[1] 任先军[1] 阴洪[1] 田永阳[1] 周长立[1] 王开见[1] 

机构地区：[1]第三军医大学附属新桥医院骨科,重庆400037

出　　处：《中国矫形外科杂志》2013年第21期2206-2211,共6页Orthopedic Journal of China

基　　金：国家自然基金青年项目(编号:31000438);国家自然科学基金面上项目(编号:81271362);军队"十二五"重点项目(编号:BWS11C040)

摘　　要：[目的]研究慢病毒载体(lentiviral vectors,LVs)介导增强型绿色荧光蛋白(enhanced green fluorescent protein,eGFP)标记大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)的可行性及对细胞生物学特性的影响。[方法]采用密度梯度离心法分离培养大鼠BMSCs,经Lenti-emGFP/hygro慢病毒载体转染和潮霉素药筛,镜下观察细胞形态和eGFP表达情况,MTT法检查转染前后细胞增殖活力,流式细胞仪检测细胞表面标记物(CD34、CD44、CD45、CD90)和eGFP,检测转染后细胞的成骨、成脂分化潜能并分别行茜素红染、油红O染色鉴定。[结果]大鼠BMSCs呈成纤维细胞样形态,漩涡样状排列,转染后细胞形态不变,并高效表达eGFP,两组细胞增殖活力一致,流式细胞仪检测显示,转染后的P3代大鼠BMSCs-eGFP的eGFP阳性率为99.97%,转染前后,两组细胞CD34和CD45阳性率低于2%,而CD44和CD90阳性率高于99%。转染后的P3代大鼠BMSCs-eGFP成骨诱导后,茜素红染色,可见呈桔红色的块状钙结节,成脂诱导后,油红O染色显示有大量红色脂质沉淀,成骨成脂诱导过程中,eGFP持续表达。[结论]Lenti-emGFP/hygro转染大鼠BMSCs并经潮霉素药筛后,eGFP可稳定高效表达,对细胞的生物学特性无明显影响,是一种较为理想的标记方法。[Objective] To study the effect of lentiviral vector- mediated transfection of rat bone marrow mesenchymal stem cells （BMSCs） with an enhanced green fluorescent protein （eGFP） gene on the phenotype, surface marker expression, and differentiation capacity of rat BMSCs. [Methods] Rat BMSCs were isolated by density- gradient eentrifugation and transfeeted using lenti - emGFP/hygro lentiviral vectors. Transfeeted cells were isolated by screening for hygromycin resistance. The morphological characteristics and eGFP expression were observed using fluorescence microscopy; proliferation rates of the transfected and untransfected BMSCs were evaluated using the MTT assay; and the expression of surface markers （CD34, CD44, CD45, and CD90） and eGFP were detected by flow cytometry. The multi - lineage differentiation potential of eGFP - transfected BMSCs in the third passage （ P3 ） was tested by induction of osteogenic and adipogenic differentiation in these cells, and detection of osteogenic and adipogenic differentiated ceils by alizarin red staining and oil red O staining, respectively. [ Results ] Rat BMSCs exhibited fibroblast - like shape and spiral arrangement in culture. The transfection of these cells with lenti - emGFP/ hygro, and subsequent screening for drug - resistance had no significant effect on their morphological characteristics and proliferation. A large percentage of the transfected rat BMSCs were found to express eGFP; the percentage of eGFP - expressing ceils in the transfeeted P3 rat BMSCs culture was 99.97%. The percentage of cells expressing CD34 and CI）45 in both transfected and untransfected BMSCs was less than 2%, while that of cells expressing CD44 and CD90 was more than 99% of the BMSCs in culture. Upon osteogenic and adipogenic induction, the eGFP- transfected P3 BMSCs showed positive alizarin red staining and oil red O staining, respectively, and stably expressed eGFP during the differentiation process. [ Conclusion] Lentiviral vector-mediated transfection of rat BMSCs with th

关 键 词：骨髓间充质干细胞 基因转染 慢病毒载体 增强型绿色荧光蛋白 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]