无血清条件下体外分离培养鼠胚胎脑组织神经干细胞  

Isolation and culture of neural stem cells from the rat embryo brain under the serum-free condition in vitro

作　　者：李明新[1,2] 阚世廉[2] 张建国[2] 

机构地区：[1]天津医科大学研究生院,天津市300070 [2]天津医院手外科,天津市300211

出　　处：《中国组织工程研究与临床康复》2009年第14期2696-2700,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景:神经干细胞的体外培养成功为治疗中枢神经系统疾病提供了新的思路,但神经干细胞的分化和功能修复机制尚不甚清楚,移植后的细胞能否与体内细胞结合以及建立起正常的神经系统突触联系急需解决。目的:在无血清条件下体外分离培养胚鼠神经干细胞,观察其生长及分化情况。设计、时间及地点:细胞学体外观察,于2007-03/2008-01在天津市环湖医院神经干细胞室完成。材料:孕14～16d的SD大鼠,由北京维通利华实验动物中心提供。方法:取孕鼠胚胎的脑海马组织,通过机械分离和胰蛋白酶消化结合法体外分离培养神经干细胞,在含有碱性成纤维细胞生长因子、表皮生长因子及B27的无血清DMEM/F12培养基中传代扩增。取原代培养7d的神经干细胞,制备单细胞悬液,接种后加入含体积分数为10%胎牛血清的DMEM培养液诱导3d。主要观察指标:倒置显微镜下观察神经干细胞的增殖分化过程,并行免疫荧光染色鉴定。通过细胞免疫化学染色检测诱导分化后的细胞类型。结果:分离培养的神经干细胞在无血清培养基中不断分裂增殖,8d左右即可形成胞体透亮、折光性好的干细胞球,悬浮生长,免疫荧光染色巢蛋白呈阳性表达。神经干细胞球诱导5d,免疫细胞化学染色后可见胶质纤维酸性蛋白及神经元特异性烯醇酶呈阳性表达的细胞。结论:体外无血清条件下,分离培养的神经干细胞生长状态良好,且具有自我更新和增殖能力,诱导后能够向神经元及星形胶质细胞方向分化。BACKGROUND： The successful culture in vitro of neural stem cells （NSCs） have provided a new ideal for treating the central nervous system disease, but the mechanism of differentiation and functional repair of NSCs is still unclear. Whether posttransplantation cells can combine with in vivo cells and establish a normal neural system deserves further studies. OBJECTIVE： To isolate NSCs from rat embryos in vitro, and to observe the growth and differentiation of NSCs under serum-free contidions. DESIGN, TIME AND SETTING： The cytology in vitro study was performed at the Room of Neural Stem Cells, Huanhu Hospital of Tianjin from March 2007 to January 2008. MATERIALS： Sprague Dawiey rats at 14-16 days pregnancy were supplied by the Animal Experimental Center of Beijing Weitonglihua. METHODS： NSCs were isolated from hippocampus of rat embryos by mechanical and chemical methods. Cells were cultured in serum-free DMEM/F12 medium, supplemented with basic fibroblast growth factor, epidermal growth factor and B27. NSCs following 7 days primary culture were made into single cell suspension, and incubated in DMEM containing 10% fetal bovine serum for 3 days. MAIN OUTCOME MEASURES： The proliferation and differentiation of NSCs were observed under the inverted microscope. Immunofluorescence staining was performed. Immunochemistry staining was used to detect the cell type following induction and differentiation. RESULTS： Isolated NSCs proliferated in serum-free medium. At day 8, NSCs formed bright body and stem cell spheres with good refraction. Immunofluorescence staining showed a positive expression of nestin. At 5 days, immunocytochemical staining demonstrated positive reactions for glial fibrillary acidic protein and neuron specific enolase. CONCLUSION： NSCs, with self-renewal and proliferative ability, were cultured in serum-free conditional medium and they can be induced to differentiate into neurons and astrocytes.

关 键 词：无血清培养 神经干细胞 分化 胚鼠 

分 类 号：R394.2[医药卫生—医学遗传学]