胚胎大鼠神经干细胞体外培养、分化与鉴定的实验研究

发布时间：2018-01-08 03:23

  本文关键词：胚胎大鼠神经干细胞体外培养、分化与鉴定的实验研究　出处：《广西医科大学》2006年硕士论文　论文类型：学位论文

  更多相关文章： 胚胎大鼠 神经干细胞 细胞培养 诱导分化

【摘要】： 目的: (1)建立简便、易行的胚胎大鼠NSCs体外分离、培养和扩增的方法。 (2)鉴定NSCs及诱导分化后的子代细胞,为NSCs的体内移植研究奠定基础。 方法: (1)从胚胎大鼠脑组织中分离NSCs,采用神经干细胞克隆悬浮法,选用DMEM/F12培养液,添加无血清培养添加剂b27,细胞生长因子(bFGF和/或EGF)进行体外扩增培养,机械分离连续传代培养,倒置相差显微镜观察细胞形态,细胞生长曲线观察NSCs的增殖能力,并采用免疫荧光细胞化学技术检测NSCs标志物Nestin的表达。 (2)观察不同浓度的bFGF、EGF对NSCs增殖的影响,选择最适合NSCs生长的细胞生长因子浓度。 (3)以多聚赖氨酸包被盖玻片置于24孔培养板中,将传代培养的NSCs接种于盖玻片上,滴加5%胎牛血清的DMEM/F12培养液,诱导分化培养7天后,用免疫荧光细胞化学技术和免疫细胞化学染色,观察GFAP、β-tubulin-Ⅲ的表达。 结果: (1)从胚胎大鼠脑组织分离的细胞在含b27及细胞生长因子bFGF、EGF的无血清DMEM/F12培养液中,能够形成大量悬浮的NSCs克隆球,并可在体外大量扩增和连续传代培养,克隆球经Nestin检测鉴定证实为NSCs。 (2)发现不同的细胞生长因子浓度对NSCs的增殖有影响,随着细胞生长因子浓度的增加,NSCs的增殖速度亦随之加快,浓度在20-30ng/ml时细胞增殖速度达到较高水平,因子浓度大于50ng/ml时细胞增殖速度反而下降。 (3)胎牛血清共培养7天后,分别做β-tubulin-Ⅲ,GFAP抗体检测,结果显示分化后的细胞部分表达神经元、星形胶质细胞的特异性抗原。
[Abstract]:Objective: To establish a simple and easy method for isolation, culture and amplification of embryonic rat NSCs in vitro. 2) Identification of NSCs and induced differentiation of progeny cells, which laid a foundation for in vivo transplantation of NSCs. Methods: 1) NSCs were isolated from embryonic rat brain tissue. Neural stem cell clone suspension method was used, DMEM/F12 culture medium was used and serum-free culture additive b27 was added. Cell growth factor bFGF and / or EGF were cultured in vitro and cultured by mechanical isolation and subculture. The morphology of cells was observed by inverted phase contrast microscope and the proliferative ability of NSCs was observed by cell growth curve. The expression of NSCs marker Nestin was detected by immunofluorescence cytochemistry. (2) to observe the effect of different concentrations of bFGF EGF on the proliferation of NSCs, and to select the concentration of cell growth factor that is most suitable for the growth of NSCs. The covered glass was coated with poly-lysine and placed in a 24-well culture plate. The NSCs cultured by passage was inoculated on the cover glass, and the DMEM/F12 medium of 5% fetal bovine serum was added to the culture medium. The expression of GFAP, 尾 -tubulin- 鈪，

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