腺相关病毒2/1型载体转染大鼠骨髓间充质干细胞的实验研究

Transfection of Bone Marrow Mesenchymal Stem Cells by Adeno-associated Virus 2/1 Vector

本研究探讨重组腺相关病毒2/1型(recombinant adeno-associated virus2/1,rAAV2/1)作为载体在不同转染复数、不同时间点转染大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSC)的效率及对其生长的影响。用含有增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因的rAAV2/1(rAAV2/1-EGFP),以感染复数(multiplicity of infection,MOI)分别为1×104、1×105、1×106转染体外培养的大鼠骨髓间充质干细胞,在3、7、14天时荧光显微镜下观察EGFP的表达,检测子代细胞的活力、增殖倍数、分化情况,以评估rAAV2/1对BMMSC存活、增殖、分化能力的影响。应用流式细胞仪检测rAAV2/1-EGFP对BMMSC的转染效率及荧光指数(fluorescence indexnumber,FI)。结果表明:转染24小时后即可观察到BMMSC发出的绿色荧光,荧光强弱不一,随着时间的延长,荧光的强度逐渐增强,7天后达到稳定的状态;在3、7、14天时不同MOIrAAV2/1-EGFP转染BMMSC的活力、增殖倍数、分化能力无明显变化(p>0.05);在同一感染复数时,7天比3天和14天比7天的增殖倍数显著增强(p<0.01)。流式细胞仪检测显示,rAAV2/1-EGFP转染BMMSC的转染率和荧光指数随着MOI增加(1×104、1×105、1×106)和培养时间(3、7、14天)的延长而有不同程度的增加(p<0.05)。结论:rAAV2/1-EGFP能有效地转染BMMSC,随转染复数的增加和短期内随转染时间的延长,转染率和荧光指数明显增加。在转染过程中rAAV2/1-EGFP对细胞的活力、生长无影响。对于改造BMMSC,rAAV2/1是一种有效的基因转移载体。

This study was aimed to iavestigate the transfection efficacy of recombinant adeno-associated virus 2/1 （rAAV2/1） on bone marrow mesenchymal stem cells （BMMSCs） at different multiplicities of infection （MOI） and time, and effect of transfection on growth of rat BMMSCs. The rat BMMSCs cultured in vitro were transfected by using rAAV2/1 with enhanced green fluorescent protein（rAAV2/1-EGFP） at MOI of 1 ×10^4、1×10^5 and 1×10^6 ; the EGFP expression was observed by fluorescent microscopy at 3,7 and 14 days. The viability, proliferation multiple, differentiation ability of daughter cells were detected for evaluating the effect of rAAV2/1 on survival, proliferation and differentiation of BMMSCs and the fluorescence index （FI） were determined by flow cytometry. The results indicated that after transfection with rAAV2/1 for 24 hours the green fluorescence in BMMSCs were observed, but also the fluorescence gradually was enhanced along with prolonging of time, and reached to steady level after 7 days; the viability, proliferation multiple, differentiation ability of BMMSCs transfected by rAAV2/1-EGFP at different MOI showed no significant changes at 3,7 and 14 days （p 〉 0.05 ）, meanwhile at same MOI the proliferation multiple obviously increased in comparison between 7 day vs 3 day and 14 days vs 7 days （p 〈0.01 ）. The flow cytometric detection showed that the transfection efficacy of rAAV2/1 -EGFP on BMMSCs and FI increased significantly as the multiplicity of infection and culture time increased（p 〈0.05）. It is concluded that rAAV2/1-EGFP is able to transfect into BMMSCs effectively, but the transfection efficiency and fluorescence index increase significantly along with increase of multiplicity of infection and culture time. rAAV2/1-EGFP do not affect viability, proliferation multiple and differentiation ability of BMMSCs. rAAV2/1 is a kind of active vector for gene transfer to reform BMMSCs.