腺病毒介导的心肌营养素1基因修饰大鼠神经干细胞

Adenovirus-mediated cardiotrophin-1 gene for modifying neural stem cells in rats

目的:以心肌营养素1基因修饰大鼠神经干细胞,为心肌营养素1基因修饰的神经干细胞移植治疗中枢神经系统损伤提供物质基础。方法:实验于2002-06/2003-06在第三军医大学野战外科研究所全军重点开放实验室进行。从W istar大鼠胚胎海马和室管膜区组织分离、培养大鼠胚胎神经干细胞,以构建纯化好的重组腺病毒-心肌营养素1和重组腺病毒-增强绿色荧光蛋白载体转染神经干细胞。相差显微镜下直接观察细胞生长存活情况,应用免疫细胞化学和免疫荧光技术检测神经干细胞及其诱导后分化细胞的特异性蛋白巢蛋白、神经微丝蛋白、胶质纤维酸性蛋白,外源性Brdu和增强绿色荧光蛋白的掺入情况,并观察心肌营养素1基因在神经干细胞中的表达。结果:①从胚胎大鼠分离的神经干细胞可在体外多次传代,经细胞增殖标记物Brdu掺入实验证明神经干细胞具有分裂增殖能力。②检测神经干细胞标志物巢蛋白、神经元标志物神经微丝蛋白和胶质细胞标志物胶质纤维酸性蛋白,证实神经干细胞具有多向分化潜能。③重组腺病毒-心肌营养素1及重组腺病毒-增强绿色荧光蛋白转染神经干细胞后应用免疫组化和荧光示踪技术,检测到神经干细胞中心肌营养素1和报告基因增强绿色荧光蛋白表达明显持续增加。结论:①实验成功地分离和培养出大鼠神经干细胞,并在体外可多次传代增殖并可诱导分化为神经元和胶质细胞。②重组腺病毒-心肌营养素1和重组腺病毒-增强绿色荧光蛋白转染神经干细胞后,外源性心肌营养素1基因和增强绿色荧光蛋白在细胞中可持续稳定表达。

AIM： To provide material foundation for the transplantation of neural stem cells （NSCs） modified by cardiotrophin-1 （CT-1） gene in the treatment of central nervous system injury with the CT-1 gene modifying NSCs in rats. METHODS： The experiment was performed at the Military Key Opened Laboratory, Institute of Battle Surgery, Third Military Medical University of Chinese PLA from June 2002 to June 2003. The embryos NSCs were isolated and cultured from embryo hippocampus and ependyma region in Wistar rats in order to establish the purified recombined adenovirus （rAd）-CT1 and rAd-enhanced green fluorescent protein （EGFP） carrier transfection into NSCs. The condition of growth and living was observed directly under the differential microscope. The NSC5 and the special nestin, neurofilament, glial fibriliary acidic protein （GFAP） exogenous Brdu of the differentiation ceils of NSCs after inducing and the mixed status of the EGFP were detected with immunocytochemical method and immunofluorescence technique, and the expression of the CT-1 gene in the NSCs was observed. RESULTS： ① The NSCs separated from embryo of rats could do many times of passage in vitro. The cell proliferation marker, Brdu mixture verified that the NSCs had the ability of splitting and proliferation. ② The NSCs marker-nestin, neuron marker-neurofilament and the glial cell marker-GFAP were detected, which indicated that the NSCs had the multipotential differentiation. （③After the transfection of rAd-CT1 and rAdEGFP into NSCs, the CT1 and the expression of reported gene EGFP in the NSCs continuously increased significantly detected with immunohistochemical method and fluorescent tracer technique. CONCLUSION： ① NSCs are isolated and cultured in the experiment successfully, and can perform passage proliferation in vitro in many times and can trans-differentiation into neurons and glial cells.② After the transfection of rAd-CT1 and rAd-EGFP into NSCs, the exogenous CT-1 gene and EGFP can express stablely in NSCs.