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 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）茜素红染茜素红染色呈色结节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 potential of P3 BMSCs-eGFP was tested by inducing their differentiation toward to osteoblasts and adipocytes, which were determined by alizarin red staining and oil red O staining respectively. Results Rat BMSCs possessed a fibroblast-like shape and whirlpool like alinement. The transfection of Lenti-emGFP/hygro and drug screening had no significant effect on the morphological characteristics and proliferation of rat BMSCs-eGFP, furthermore, eGFP expressed in rat BMSCs-eGFP. Positive rate of eGFP in P3 rat BMSCs-eGFP was 99.97%, and positive rate of CD34 and CD45 in both transfected and untransfected BMSCs was less than 2%, while CD44 and CD 90 was more than 99%. After osteogenic and adipogenic inducing, the P3 BMSCs-eGFP showed positive