大鼠胎脑神经干细胞的分离培养及电穿孔转染  

The isolating culture of neural stem cells from rat＇s fetus brain and transfection by electroporation

作　　者：刘春江[1] 尹飞[1] 郑湘榕[1] 张珊珊[1] 谭洁璐[1] 

机构地区：[1]中南大学湘雅医院儿科,长沙410008

出　　处：《中华医学杂志》2009年第42期3007-3011,共5页National Medical Journal of China

基　　金：国家自然科学基金（30772341）

摘　　要：目的分离培养大鼠胎脑神经干细胞（NSC），研究电穿孔技术在NSC中的转染效率。方法从SD胎鼠脑组织中分离、培养和扩增NSC，利用免疫荧光组织化学技术对NSC及其分化细胞进行鉴定。使用电穿孔技术将质粒pEGFP．N1转染人NSC，利用其表达的绿色荧光蛋白（GFP）作为转染成功的报告物或标记物，在荧光显微镜下根据发出绿色荧光的NSC数量，计算出转染效率。结果从SD胎鼠脑组织中分离的细胞在体外可长时间自我增殖，原代及传代克隆细胞均表达NSC的特异性抗原巢蛋白，且诱导分化后可表达星形胶质细胞的特异性抗原-胶质纤维酸性蛋白（GFAP）和神经元的特异性抗原．神经元特异性烯醇化酶（NSE）。用电穿孔技术转染神经干细胞，其效率为17．9％～69．1％，平均30．5％。结论本实验成功分离出具有自我增殖和多向分化潜能的胎鼠NSC，并证实电穿孔技术可对其进行高效转染，为进一步研究NSC的转基因治疗提供实验基础。Objective To isolate and culture the neural stem cells （NSC） from rat＇s fetus brain, to study the transfection efficacy of NSC using electroporation. Methods We isolated, cultured and amplified NSC from the brain of SD fetal rats. NSC and differentiated cells were identified using immunofluorescent histochemical methods. Using green fluorescence protein（GFP） as the marker, pEGFP-N1 was transfected into NSC using electroporation, and the tranfection rate was calculated by counting the NSCs with green fluorescent. Results The cells isolated from brain tissue of fetal rats can proliferate for long time, both primary and passage culture of NSC can express specific antigen of NSC-Nestin, and after induced differentiation, the cells can express specific antigen of astrocytes-Glial fibrillary acidic protein （GFAP） and specific antigen of neurons-Neuron-specific enolase （ NSE ）. The tranfection rates of electroporation were 17.9% -69. 1%, the average was 30.5%. Conclusion Isolated NSC which had the features of selfproliferation and differentiation potential from brain tissue of SD fetal rats, confirmed that electroporation was an efficient transfection method for NSC, provided experimental basis for gene therapy in the future.

关 键 词：神经干细胞 分离培养 电穿孔 转染 

分 类 号：R686[医药卫生—骨科学]