绿色荧光蛋白转染大鼠骨髓间充质干细胞及体外向神经元样细胞的分化  

Differentiation of rat bone marrow mesenchymal stem cells transfected with green fluorescent protein into neuron-like cells in vitro

作　　者：袁维[1] 路磊[2] 

机构地区：[1]上海市第八人民医院骨科,上海市200235 [2]中国医科大学华宇医院骨科,浙江省绍兴市312030

出　　处：《中国组织工程研究与临床康复》2008年第47期9253-9256,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：辽宁省博士启动资助基金(20021031)~~

摘　　要：背景:成年骨髓间充质干细胞诱导后在体外可分化为神经元样细胞,利用细胞转染技术让其表达绿色荧光蛋白,便于在动物模型体内跟踪观察。目的:观察增强型绿色荧光蛋白转染的骨髓间充质干细胞体外分化为神经元样细胞的能力,拟为增强型绿色荧光蛋白基因作标志物对外源性骨髓间充质干细胞进行体内追踪做准备。设计、时间及地点:细胞学体外观察,于2006-06/2007-06在中国医科大学医学分子生物学国家重点实验室完成。材料:清洁级成年Wistar大鼠32只,由中国医科大学实验动物部提供。增强型绿色荧光蛋白质粒为Sigma公司产品。方法:取大鼠两侧股骨及胫骨,Percoll密度梯度离心+贴壁法体外分离培养骨髓间充质干细胞,达90%融合后消化传代扩增。由脂质体介导,将含有增强型绿色荧光蛋白质粒cDNA转入骨髓间充质干细胞,经G418筛选后,按0.4×109L-1接种,加入含FBS、碱性成纤维细胞生长因子的DMEM预诱导24h,更换培养液后再以含BHA、DMSO的无血清DMEM诱导5h～6d。主要观察指标:荧光显微镜观察细胞转染情况,免疫细胞化学染色检测转染细胞诱导后神经元表面抗原的表达。结果:携带增强型绿色荧光蛋白基因的骨髓间充质干细胞生长旺盛,转染24h后在细胞核中就可见弥散绿色荧光,转染48h后绿色荧光更加集中,在细胞核中聚集成团块、颗粒状,体外培养3个月后仍能够高水平表达增强型绿色荧光蛋白。与未转染细胞比较,转染细胞诱导后其神经元特异性烯醇化酶、神经丝蛋白、巢蛋白阳性率均无明显变化(P>0.05),胶质纤维酸性蛋白呈阴性表达。结论:骨髓间充质干细胞体内能稳定、高效和持久地表达增强型绿色荧光蛋白,转染后生物学性状未发生改变,体外在细胞因子和氧化剂的诱导作用下仍可以分化为神经元样细胞。BACKGROUND： Adult bone marrow mesenchymal stem cells （BMSCs） after induction can differentiate into neuron-like cells in vitro. Using cell transfection, BMSCs express green fluorescent protein, and are convenient for trace observation in animal models. OBJECTIVE： To explore the ability of transdifferentiation of BMSCs transfected with enhanced green fluorescent protein into neuron-like cells in vitro, and to offer references for the application of tracing BMSCs in vitro with enhanced green fluorescent protein transplanted into animal models in vivo. DESIGN, TIME AND SETTING： The cytology in vitro observation was performed at the National Key Laboratory of Medical Molecular Biology, China Medical University from June 2006 to June 2007. MATERIALS： Thirty-two clean adult Wistar rats were supplied by Department of Experimental Animal of China Medical University. Enhanced green fluorescent protein plasmid was obtained from Sigma.USA. METHODS： BMSCs were harvested from the rat bilateral femur and tibia using Percoll density gradient centrifugation plus adherence. When 90% cells were confluent, BMSCs were digested and cultured. Enhanced green fluorescent protein plasmid cDNA was transfected into BMSCs mediated by liposome. After G418 screening, BMSCs at a density of 0.4×10^9 L^-1 were incubated in DMEM, supplemented with fetal bovine serum and basic fibroblast growth factor for 24 hours. After changing medium, BMSCs were incubated in serum-free DMEM containing BHA and dimethyl sulphoxide for 5 hours to 6 days. MAIN OUTCOME MEASURES： Cell transfection was observed with a fluorescence microscope. Expression of neuron surface antigen after induction was detected using immunocytochemical staining. RESULTS： BMSCs with enhanced green fluorescent protein expression greatly grew. At 24 hours after transfection, green fluorescence was found in the nuclei. At 48 hours, the green fluorescence was more, in mass, granule-shape. At 3 months, enhanced green fluorescent protein was still highly expressed. Compared wi

关 键 词：间充质干细胞 增强型绿色荧光蛋白 转染 诱导分化 

分 类 号：R394.2[医药卫生—医学遗传学]