大鼠胚胎中脑腹侧来源神经干细胞的分离和培养  

Isolation and expansion of rat embryonic ventral mesencephalic neural stem cells

作　　者：陈孝祥[1] 宋晓斌[1] 王向鹏[1] 周磊[2] 徐蛟天 林海[1] 杨智勇[1] 邓兴力[1] 

机构地区：[1]昆明医科大学第一附属医院神经外科,昆明650032 [2]昆明医科大学昆明医科大学分子临床医学研究院暨云南省干细胞和再生医学重点实验室,昆明650032

出　　处：《神经解剖学杂志》2017年第1期75-79,共5页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金(81360197,81241126);云南省教育厅科学研究基金项目(2013C227);云南省科技厅-昆明医学院联合专项(2014FB041)

摘　　要：目的:体外培养大鼠胚胎中脑腹侧来源神经干细胞(NSCs),观察其生长及诱导分化培养特点。方法:取孕12.5~14.5 d的SD大鼠,采用机械分离法获取中脑腹侧组织,剪碎并吹打成单细胞悬液后,以2×10~5个细胞/ml的密度接种到无血清NSCs培养基中以神经球法培养。培养神经球5~7 d后,按原密度进行传代培养,并取第三代培养的神经球用含10%胎牛血清(FBS)分化培养液分化培养,分化培养7 d后行免疫细胞化学鉴定。结果:神经球呈巢蛋白(nestin)阳性,诱导分化后的细胞作分别呈微管蛋白(βIII-Tubulin)、胶质纤维酸性蛋白(GFAP)阳性。结论:经神经球法培养得到的大鼠胚胎中脑腹侧来源神经干细胞可自我增值,并可诱导分化成神经元和胶质细胞。Objective;To isolate and culture the rat embryonic mesencephalic neural stem cells（mNSCs） in vitro and to observe the characteristics of the growth and proliferation of mNSCs.Methods：The ventral mesencephalic tissue was harvested from embryonic day 12.5 ~ 14.5 using microdissection.Then,the single cell suspension was achieved by mechanical dissociation using 1 ml pipette tip.The cells were plated at the density of 2 × 10~5 cells/mL in complete NSC medium without serum.After 5 ~7 days in vitro culture,the first-generation of neurospheres was formed.For in vitro differentiation,7-day-old third-generation neurospheres were induced in the same medium,with the exception of EGF and bFGF were replaced with 10%foetal bovine serum.The neurospheres and the differentiated cells were identified by Nestin,βⅢ-Tubulin and GFAP.Results：The neurospheres are identified as nestin positive and meanwhile the differentiated cells showed p-tubulin Ⅲ and GFAP immunopositive.Conclusion：The neural stem cells derived from embryonic mesencephalic has the ability to proliferate and can be induced to differentiate into neurons and glia.

关 键 词：神经干细胞 神经球 胚胎中脑腹侧组织 大鼠 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]