星形胶质细胞条件培养液体外诱导胎鼠室管膜前下区神经干细胞向多巴胺能神经元的分化  

Differentiation of neural stem cells from the anterior subventricular zone into dopaminergic neurons induced by astrocyte-conditioned medium in vitro

作　　者：辛志成[1] 周政[1] 严稽文[1] 

机构地区：[1]解放军第三军医大学新桥医院神经外科,重庆市400037

出　　处：《中国组织工程研究与临床康复》2009年第6期1040-1044,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:在神经干细胞移植治疗神经系统退行性变及修复神经系统功能损伤过程中,有效的神经干细胞体外增殖与多巴胺能神经元的定向诱导分化尤为关键。目的:以星形胶质细胞条件培养液为诱导剂,观察胎鼠室管膜前下区神经干细胞体外向多巴胺能神经元的分化。设计、时间及地点:细胞学体外对照观察,于2006-12/2007-08在解放军第三军医大学新桥医院实验中心完成。材料:清洁级新生2d龄KM小鼠15只,孕16dWistar大鼠40只,均由解放军第三军医大学实验动物中心提供。方法:采用差速黏附法和振荡分离法纯化培养KM小鼠星形胶质细胞,收集传至第3代、培养5d的星形胶质细胞条件培养液,-20℃冻存待用。体外分离Wistar胎鼠室管膜前下区神经干细胞,加入含B27和碱性成纤维细胞生长因子的DMEM/F12无血清培养基进行原代培养,传至第3代后按0.5×108L-1密度接种,设立2组:对照组单纯加入含体积分数0.1为胎牛血清的DMEM/F12培养基予以自然分化,实验组加入已制备的星形胶质细胞条件培养液进行诱导分化。主要观察指标:免疫细胞化学染色鉴定胎鼠室管膜前下区神经干细胞,流式细胞仪检测胎鼠室管膜前下区神经干细胞向多巴胺能神经元分化的阳性率。结果:培养的神经球细胞表达巢蛋白,可分化为神经元特异性烯醇化酶、胶质纤维酸性蛋白阳性细胞。诱导分化7d后,实验组可见酪氨酸羟化酶阳性细胞,胞体呈圆形或椭圆形,酪氨酸羟化酶位于胞质及突起中;对照组酪氨酸羟化酶阳性细胞在数量、细胞成熟形态上均未达到实验组水平。实验组酪氨酸羟化酶阳性细胞分化率明显高于对照组(t=35.296,P<0.01)。结论:在体外星形胶质细胞条件培养液可显著促进胎鼠室管膜前下区神经干细胞向多巴胺能神经元分化。BACKGROUND： Effective proliferation in vitro of neural stem cells and directional induction and differentiation of dopaminergic neurons play a key role in neural stem cell transplantation for treatment of degeneration of the nervous system and repair of injured nervous system. OBJECTIVE： To explore the differentiation of neural stem cells from anterior subventricular zone into dopaminergic neurons in ast rocyte-conditioned medium. DESIGN, TIME AND SETTING： The in vitro controlled cytology experiment was performed at the Experimental Center of Xinqiao Hospital of Third Military Medical University of Chinese PLA from December 2006 to August 2007. MATERIALS： A total of 15 neonatal KM mice aged 2 days and 40 pregnant 16 days Wistar rats were provided by the Experiment Animal Center of Third Military Medical University of Chinese PLA. METHODS： Astrocytcs of KM mice were isolated and purified by a standard shaking method and differential adhesion. Astrocyte-conditioned medium was collected when astrocytcs at passage 3 were cultured for 5 days maintained at -20℃ for use. Neural stem cells from the anterior subventricular zone of Wistar rats were isolated and cultured in serum-free DMEM/F12, supplemented with B27 and basic fibroblast growth factor for primary culture. Neural stem cells at passage 3 were collected and incubated at 0.5×10^8/L. Cells in the control group were incubated in the DMEM/F12 medium containing fetal bovine serum of 0.1 volume fraction. Cells in the experimental group were incubated in the astrocyte-conditioned medium. MAIN OUTCOME MEASURES： Neural stem cells in the anterior subventricular zone were determined by the immunocytochemical technique. Positive rate of the neural stem cell differentiation into dopaminergic neurons in the anterior subventricular zone was detected by the flow cytometry. RESULTS： Cells in neurospheres expressed nestin and differentiated into neuro-specific enolase and glial fibrillary acidic protein. Tyroxine hydroxylase positive cells were found in the expe

关 键 词：神经干细胞 星形胶质细胞条件培养液 多巴胺能神经元 

分 类 号：R394.2[医药卫生—医学遗传学]