人胚神经干细胞分离培养与鉴定方法  

Isolation,culture and identification of human embryo neural stem cells

作　　者：赵荣山[1] 李秀芹[1] 郝京生[1] 董玉环[1] 赵亚朴[2] 王本旭[1] 

机构地区：[1]北京军区联勤部军事医学研究所微生物检验科,石家庄050081 [2]白求恩军医学院病原教研室,石家庄050081

出　　处：《华北国防医药》2004年第2期80-82,F003,共4页Medical Journal of Beijing Military Region

摘　　要：目的 :建立神经干细胞 (NSCs)实验室分离、培养方法 ,为NSCs移植提供细胞源。方法 :取 4月龄 (± 15天 )正常孕妇水囊引产胎儿大脑纹状体区组织分离神经干细胞 ,培养于含碱性成纤维细胞生长因子 (bFGF)和B2 7的无血清培养基 ;同时利用单细胞克隆技术进行连续传代培养 ;利用形态学观察和免疫荧光技术检测神经上皮干细胞蛋白Nestin抗原的表达来鉴定神经干细胞。结果 :体外培养的干细胞在bFGF和B2 7培养基中可不断增殖形成细胞球 ,并出现少量细胞分化。单细胞培养 4天即开始分裂 ,同时伴有个别细胞分化 ;出现大量神经球一般为 15天 ;分化的细胞在 3 0天开始分解 ,5 0天左右基本消失。细胞培养 3个月 (每月补液 1次 )仍具有分裂增殖能力。单细胞克隆可连续传代 10次 ,仍具增殖分化能力。液氮冻存 6个月的细胞复苏后仍具增殖分化能力。单细胞克隆传代增殖的细胞球经Nestin免疫荧光鉴定 ,呈阳性结果 ,证实其胚胎源性。结论 :采用bFGF和B2 7的无血清培养基能促进神经干细胞连续稳定增殖 ,并有少量分化 ,细胞体外培养 3个月、克隆连续传代Objective:To establish neural stem cells(NSCs) isolation and culture in vitro in order to supply the cells for the transplant of NSCs.Methods:The striatum brain tissue was isolated from human embryo (clinically aborted, 4 months ±15 d of pregnancy) and cultured with serum free culture medium including bFGF and B27;A Single clone was cultured in consecutive generations by single cell cloning technique.Nestin antigen expression was detected as the marker of NSCs with morphological and immunofluorescence techniques.Results:Human neural stem cells cultured in vitro in serum free medium with bFGF and B27 could constantly proliferate to form neurospheres and some of them showed a certern degree of differentiation.On the 4th day,division of NSCs started at the same time a few of the cells differentiated.A great number of neurospheres appeared on the 15th day;on the 30th dayd,the differentiated cells started to break down;around the 50th day,most of the cells disappeared.After 3 months of culture (the medium was refreshed monthly),the ability of devision and proliferation still remained in NSCs.The cells from monoclonal cell could still proliferate and differentiate after 10 generations.When having been frosen in liquid nitrogen for 6 months,the cells were still able to proliferate and devide after anabiosis.The expression of Nestin was found in neurospheres and this confirmed the characters of embryo source.Conclusions:Serum free medium with bFGF and B27 could promote a constant proliferation of human NSCs;a few of NSCs differentiated in vitro.The features of NSCs still remained after 3 months of culture and 10 generation of the cell cloning.

关 键 词：神经干细胞 细胞分离 细胞培养 鉴定 胚胎 单细胞克隆技术 

分 类 号：Q813.11[生物学—生物工程]