南京大学学报(自然科学版) ›› 2014, Vol. 50 ›› Issue (6): 883–889.

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慢病毒介导的GFP转染对小鼠骨髓间充质干细胞表型、增殖和分化能力的影响

李燕12 ，陆伟2 竺丽梅2 邵燕2 陈诚2 刘巧2 韩晓冬1   

  • 出版日期:2014-11-24 发布日期:2014-11-24
  • 作者简介:（1.福建农林大学管理学院旅游学院，福州，350002； 2.福建农林大学食品科学学院，福州，350002；
    3.福建师范大学数学与计算机科学学院，福州，350108；4.兰州理工大学材料科学与工程学院，兰州，730050）
  • 基金资助:
    国家自然科学基金（81301448，81301448，3120040），江苏省疾病预防控制中心重点人才项目（JKRC20110029）

Mouse bone marrow?mesenchymal?stem cells?(MSCs)transfected with?GFP gene by Lentiviral?vector

Li Yan 1,2, Lu Wei 2, Zhu Limei2, Shao Yan2, Chen Cheng2, Liu Qiao2,Han Xiaodong1*   

  • Online:2014-11-24 Published:2014-11-24
  • About author:(1. Medical School, Nanjing University, Nanjing, 210093, China; 2. Department of Chronic Communicable Disease, Jiangsu Provincial Center for Disease Prevention and Control, Nanjing, 210009, China)

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摘要： 运用慢病毒（Lentivirus）载体构建绿色荧光蛋白（GFP）在小鼠骨髓间充质干细胞MSCs）中的转染体系，检测转染后MSCs的增殖能力，并诱导其向成肌细胞分化，检测此转染体系对MSCs分化能力的影响。骨髓细胞悬液破红后贴壁法培养MSCs，采用流式细胞术鉴定细胞表面标记，鉴定MSCs纯度；Lentivirus-GFP 以1、10、50和100 的感染复数（MOI）转染MSCs，作用48 h后流式细胞术及免疫荧光法检测转染效率和表达的荧光强度；MTT 法检测转染后MSCs 的细胞活力。诱导MSCs-GFP向成肌细胞分化，蛋白印记法（WB）检测成肌细胞特异性蛋白desmin和α-SMA的表达。流式细胞术检测结果显示，培养的MSCs表达 CD44, CD90和 CD105，不表达CD34, CD45和CD188，符合干细胞特性。MOI 为1、10、50和100的转染效率分别为23.45%、93.51%、95.44%和95.55%，MOI为10时，转染效率较高，且对MSCs活力无明显影响。MSCs-GFP体外经成肌细胞诱导分化后，表达特异性抗原desmin和α-SMA。本研究成功构建了小鼠MSCs的Lentivirus-GFP转染体系，有效标记了MSCs细胞，且此标记对MSCs的细胞生物学特性无明显影响。

Abstract: The object of this study is to construct the green fluorescent?protein ?(GFP) transfection?system by?lentiviral vector?in mouse bone marrow mesenchymal?stem cells?(MSCs)?in vitro. We detected the biological characteristics of mesenchymal?stem cells?(MSCs), inclouding cell phenotype,?proliferation ability and differentiation ability of mesenchymal?stem cells?after lentiviral –GFP transfection. Then we observed the myoblasts differentiation ability of mesenchymal?stem cells?(MSCs) after we?confirmed?the expression of green fluorescent?protein. Bone marrow?cell suspensions?from mice bone were adherent?cultured after erythrocyte broken. We detect the?cell surface marker (CD44,?CD90?, CD105,,CD34,?CD45?and CD188.)?of MSCs to?identify the?purity of mesenchymal?stem cells by flow cytometry analysis. We transfected mesenchymal?stem cells (MSCs) with Lentivirus-GFP?for 48 hours, with?the multiplicity of infection (MOI) of 1,?10,?50?and 100, respectively. The transfection efficiency and fluorescence expression?were detected?by both flow cytometry?and ?immunofluorescence?; we evaluated the cell viability by the 3-（4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideMTT) test after lentiviral –GFP transfection. MSCs-GFP were induced into myoblasts after the transfection were completed, the expression?of myoblast?specific protein?desmin and?α –SMA were detected by western blot?(WB) method. The results?of flow cytometry?analysis shown: In accordance with?the characteristics of stem cells surface?markers, our cultured mesenchymal?stem cells?(MSCs)?expressed CD44,?CD90?and CD105,?but not express CD34,?CD45?and CD188. The?transfection efficiency?were 23.45%,?93.51%,?95.44% and?95.55%, while the transfect MOI were 1,?10,?50 and?100, respectively. At transfect MOI 10,?the transfection efficiency of mesenchymal?stem cells?(MSCs) was the best,?and there was no obvious?effect on?cell activity observed. After myoblast?differentiation in vitro, the MSCs-GFP?could express desmin?and?α–SMA. In this study, we constructed the green fluorescent?protein ?(GFP) transfection?system by?lentiviral vector?, this transfect system?could labeled the mouse bone marrow mesenchymal?stem cells?(MSCs) successfully ?in vitro. This transfect system?has no obvious effect on cell?biological?characteristics?of?mice MSCs, inclouding cell phenotype,?proliferation ability and differentiation ability of mesenchymal?stem cells?

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