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

Co-expression of both green fluorescent protein and myogenic regulatory factors in bone marrow-derived mesenchymal stem cells

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.

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）. Ad-GFP was amplified and identified to transfect MSCs. Expressions of both MyoD and Myogenin were detected in transfected MSCs by RT-PCR. Differentiation of MSCs was induced by cocultured with C2C12 myoblasts, and MyoD expression was identified by （IF） RESULTS： FCM showed that CDllb and CD45 were negative, CD29 and CD44 were positive in MSCs surface antigens. With the increasing of Ad-GFP 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 RT-PCR; and MyoD protein can be found in induced MSCs by IF. CONCLUSION： MSCs can be effectively transfected by Ad-GFP and express GFP; and activation of intrinsic myogenic regulatory factors will enhance the myogenic differentiation of MSCs.