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.docVIP

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基金项目： 基金项目：国家自然基金青年项目，国家自然科学基金面上项目，军队“十二五”重点项目（BWS11C040） 作者简介：蒋涛，男，主治医师，讲师，博士研究生，研究方向：脊柱外科，电话 E-mail: fromcq2000@。 *通讯作者：任先军，电话：023E-mail: ren_xianjun@ 蒋涛，任先军，阴洪，田永阳，周长立，王开见 （400037 重庆，第三军医大学附属新桥医院骨科） [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。 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 The study on the biological characteristics of rat BMSCs transfected with lentiviral vectors mediated eGFP Jiang Tao, Ren Xianjun, Yin Hong, Tian Yongyang, Zhou Changli, Wang Kaijian (Department of Orthopedics, Xinqiao Hospital, The Third Military Medical University, Chongqing 400037, China) [abstract] Objective To investigate the feasibility of transfection of rat bone marrow mesenchymal stem cells (BMSCs) with lentiviral vectors mediated enhanced green fluorescent protein (eGFP) gene and observe the influence for biological characteristics of BMSCs. Methods Rat BMSCs were isolated by density gradient centrifugation, and transfected by Lenti-emGFP/hygro lentiviral vectors followed by dug screening of hygromycin. The morphological characteristics and eGFP expression of BMSCs were observed by fluorescent microscope, the proliferation of the transfected and untransfected BMSCs were evaluated by MTT, and their surface markers （CD34、CD44、CD45、CD90）and eGFP were examined by flow cytometry. And then, the multi-differentiation pote