小鼠胚胎神经干细胞的体外生长特性和相关基因的表达  

In vitro growth characteristics and related gene expression of neural stem cells from embryo of mouse

作　　者：山爱景[1] 郭国庆[1] 孙艳[2] 沈伟哉[1] 

机构地区：[1]暨南大学医学院解剖教研室,广东省广州市510630 [2]暨南大学血液病研究所,广东省广州市510630

出　　处：《中国临床康复》2006年第5期19-21,i0002,共4页Chinese Journal of Clinical Rehabilitation

基　　金：广东省自然科学基金团队项目(039213);广州市科技计划项目(2004J1-C0241);广东省中医药管理局项目(401040)资助~~

摘　　要：目的:观察小鼠胚胎神经干细胞的体外生长特性并检测其相关基因的表达。方法:实验于2005-02/06在暨南大学医学院中心实验室进行。①取妊娠13.5d昆明小鼠分离胚胎大脑中的神经干细胞,用添加碱性成纤维细胞生长因子的无血清培养基进行体外培养,取培养3d的神经球接种于多聚赖氨酸包被的培养瓶,用含血清的培养基诱导分化。②免疫细胞化学检测细胞表面抗原。③四甲基偶氮唑盐法绘制生长曲线。④流式细胞术检测细胞周期。⑤反转录-聚合酶链反应和免疫细胞化学技术对神经干细胞进行鉴定。结果:①胚胎神经干细胞及其分化细胞的抗原表达:血清诱导8h,分化细胞中(8.9±5.6)%细胞呈Tubulin阳性,(87.5±7.4)%细胞胞浆呈胶质纤维酸性蛋白阳性表达。未诱导细胞近100%呈巢蛋白阳性,仅有极少数细胞Tubulin阳性或胶质纤维酸性蛋白阳性。②胚胎神经干细胞的细胞周期:从小鼠胚胎大脑获取的神经干细胞3d左右进入指数生长期;原代培养细胞83.8%处于静止期/DNA合成前期。③原代与传代细胞均表达巢蛋白mRNA,聚集形成的神经球巢蛋白阳性。血清诱导4h胚胎神经干细胞开始转录神经元相关基因Tau和胶质细胞相关基因胶质纤维酸性蛋白。结论:从小鼠胚胎大脑可成功分离获得大量胚胎神经干细胞,并在含碱性成纤维细胞生长因子的条件培养基中迅速增殖,血清诱导下分化生成神经元和星型胶质细胞。AIM： To observe the in vitro growth characteristics of neural stem cells from embryo of mouse, and detect the expression of related gene. METHODS： The experiment was carried out in the central laboratory of Medical College, Jinan University between February and June 2005.① Neural stem cells were isolated from cerebra of mouse embryos, which was pregnant for 13.5 days. The harvested cells were cultured in serum-free media containing basic fibroblast growth factor （bFGF）. The neurospheres cultured for 3 days were inoculated to the cultured bottle coated by poly-L-Lycine, and then induced to differentiate in culture media containing serum. ② The antigen on the cell surface was detected with immunocytochemistry. ③ The growth curve was drawn by the method of methyl-thiazol-tetrazolium （MTF）. ④ They cell eyele was detected with flow cytometry. ⑤ Neural stem cells were identified with reverse transcription-polymerase chain reaction （RT-PCR） and immunocytochemistry. RESULTS： ① Expression of the neural stem cells from embryo and the antigen of the differentiated cells： At 8 hours of serum induction, （8.9±5.6）% of the differentiated cells showed Tubulin positive, and （87.5 ±7.4）% cytoplasm showed positive expression of glial fibriliary acidic protein. About 100% of the cells that were not induced appeared positive nidogen, only extremely a few cells had Tubulin positive or positive expression of glial fibriliary acidic protein. ② Cell cycle of the neural stem cells from embryo： The nerve stem cells isolated from the embryonic cerebra entered the exponential growth phase at about 3 days. 83.8% of the primary cuhured cells stayed in the G0/G1 phases.③ Cells in primary culture and passaged expressed nestin mRNA and protein in cytoplasm, After induced by serum for 4 hours, nerve stem cells began to express neuron-specific gene Tau and glial cells-specific gene glial fibriliary acidic protein. CONCLUSION： Neural stem cells can be successfully isolated from the cerebra of mouse

关 键 词：干细胞/细胞学 细胞分化 脑/胚胎学 细胞 培养的 流式细胞术 逆转录聚合酶链反应 

分 类 号：R394.2[医药卫生—医学遗传学]