绿色荧光蛋白和成肌调节因子在骨髓基质干细胞中的共表达

绿色荧光蛋白和成肌调节因子在骨髓基质干细胞中的共表达 作者：李美山，张成，陈松林，于美娟，张雅妮，王淑辉，熊符 【关键词】 腺病毒 Coexpression of both green fluorescent protein and myogenic regulatory factors in bone marrowderived mesenchymal stem cells 　　【Abstract】 AIM: To investigate the transfection of bone marrowderived mesenchymal stem cells (MSCs) by recombinant adenovirus (Ad), and the expressions of both green fluorescent protein (GFP) and myogenic regulatory factors, which are MyoD and Myogenin. METHODS: Rat MSCs were separated, cultured and expanded in vitro, and taken for identification of surface antigens by flow cytometry (FCM). AdGFP was amplified and identified to transfect MSCs. Expressions of both MyoD and Myogenin were detected in transfected MSCs by RTPCR. Differentiation of MSCs was induced by cocultured with C2C12 myoblasts, and MyoD expression was identified by immunofluorescence (IF). RESULTS: FCM showed that CD11b and CD45 were negative, CD29 and CD44 were positive in MSCs surface antigens. With the increasing of AdGFP multiple of infection (MOI), transfection rate were also increased. When MOI was over 100, cytopathic effect appears on MSCs. Expressions of both MyoD and Myogenin in transfected MSCs were approved by RTPCR; and MyoD protein can be found in induced MSCs by IF. CONCLUSION: MSCs can be effectively transfected by AdGFP and express GFP; and activation of intrinsic myogenic regulatory factors will enhance the myogenic differentiation of MSCs. 　　【Keywords】 mesenchymal stem cells; adenovirus; green fluorescent protein; C2C12; MyoD; Myogenin 　　【摘要】 目的：研究重组腺病毒（Ad）对骨髓基质干细胞（MSCs）的转染，以及绿色荧光蛋白（GFP）和成肌调节因子MyoD和Myogenin在MSCs中的表达. 方法：用差速贴壁法体外培养大鼠MSCs，并用流式细胞仪（FCM）检测其表面标志；对构建有绿色荧光蛋白的重组腺病毒（AdGFP）进行扩增和鉴定，并转染MSCs；用RTPCR检测MyoD和Myogenin在转染后MSCs中的表达；将MSCs与C2C12成肌细胞共培养，诱导前者的分化，并用免疫荧光检测MyoD的表达. 结果：FCM检测证实，在MSCs的表面标志中CD11b和CD45阴性，而CD29和CD44阳性；随着AdGFP感染复数（MOI）的增加，转染效率也相应提高，但在MOI大于100后，MSCs开始出现病变；RTPCR结果提示MyoD和Myogenin在转染后的MSCs中有表达；免疫荧光检测证实诱导后的MSCs可以表达MyoD蛋白. 结论：MSCs可以被AdGFP有效转染而表达GFP；同时内源性成肌调节因子的激活，可以促进MSCs的成肌分化. 　　【关键词】 基质干细胞；腺病毒；绿色荧光蛋白；C2C12；MyoD；Myogenin 　　0引言 　　骨髓基质干细胞（mesenchymal stem cells, MSCs）具有向成骨、脂肪、神经以及肌肉等多种类型细胞分化的潜能，其易于分离和扩增的特性，使其成为细胞治疗的一种很有前景的治疗手段［1-3］. 为促使MSCs更好地定向分化，对其进行外源性基因修饰和/或内源性基因的激活，成为目前亟待解决的问题. 我们通过构建有绿色荧光蛋白（green fluorescent protein, GFP）的５型重组腺病毒（AdGFP）转染大鼠的MSCs，并与C2C12成肌细胞共培养，通过激活内源性成肌调节因子，启动MSCs的成肌分化. 　　1材料和方法 　　1.1材料DMEM和1640培养基（Gibco公司）、胎牛血清（杭州四季青公司）、新生牛血清（Hayclone公司）、FITC荧光标记小鼠抗大鼠抗体（PharMingen and Serotec公司）、人胚肾293细胞和AdGFP（中山大学黄文林教授惠赠）、C2C12细胞（中山大学潘秋