胚胎大鼠神经干细胞体外培养鉴定及诱导其分化为类毛细胞的研究

发布时间：2018-01-05 15:42

本文关键词：胚胎大鼠神经干细胞体外培养鉴定及诱导其分化为类毛细胞的研究　出处：《华中科技大学》2008年硕士论文　论文类型：学位论文

【摘要】： 第一部分无血清培养条件下神经干细胞的生长及分化的研究目的:探讨体外无血清条件下培养胚鼠神经干细胞(NSCs),观察其生长及分化情况. 方法:取孕16-18天SD系大鼠的胚胎海马组织,在含EGF,bFGF,B27的DMEM/F12培养基中培养,电子显微镜下观察其增殖分化过程,并通过免疫荧光方法鉴定分化后的细胞类型. 结果:NSCs在无血清培养基中生长旺盛,8天左右就可以形成胞体透亮、折光性好的干细胞球,分化后的细胞显示NSE,GFAP免疫阳性. 结论:无血清培养条件下NSCs生长良好,在含血清的培养基中可以分化为神经元和星形胶质细胞. 第二部分神经干细胞与体外培养的乳鼠基底膜上清液共培养分化为类毛细胞的研究目的:探讨体外诱导胚胎大鼠神经干细胞(neural stem cells,NSCs)分化为类毛细胞的实验研究。方法:从SD系胚鼠大脑分离NSCs,在无血清培养基中培养后将其放到含鼠尾胶原包被的盖玻片的6孔培养板中培养,然后将乳鼠基底膜取出体外培养后汲取其上清液与NSCs一起培养,14-21天后通过免疫荧光和免疫组化法检测毛细胞标志物myosin VIIa和calretinin,并计算分化比例。结果: NSCs分化后的细胞约有15.4%呈myosin VIIa免疫荧光阳性,21.7%呈免疫组化calretinin阳性。结论:体外培养的乳鼠基底膜上清液能诱导NSCs分化为类毛细胞。第三部分神经干细胞与乳鼠基底膜共培养后分化为类毛细胞的研究目的:探讨体外诱导神经干细胞(neural stem cells,NSCs)分化为类毛细胞的实验研究。方法:从SD系胚鼠大脑分离NSCs,在无血清培养基中培养。取出生3天内乳鼠基底膜与NSCs一起培养,14-21天后通过免疫荧光和免疫组化法检测毛细胞标志物myosin VIIa和calretinin,并计算分化比例。结果: NSCs分化后的细胞约有17.1%呈myosin VIIa免疫荧光阳性,27.4%呈免疫组化calretinin阳性。结论:乳鼠基底膜与NSCs共培养时能引导其朝毛细胞方向分化。
[Abstract]:Part one: growth and differentiation of neural stem cells in serum-free culture Aim: to investigate the growth and differentiation of embryonic rat neural stem cells (NSCs) cultured in serum-free medium in vitro. Methods: the embryonic hippocampal tissues of SD rats were collected from 16-18 days gestation and cultured in DMEM/F12 medium containing EGFN bFGFFGFB27. The process of proliferation and differentiation was observed under electron microscope, and the differentiated cells were identified by immunofluorescence. Results in serum-free medium, stem cell spheres with bright cell body and good refraction could be formed after 8 days of vigorous growth. The differentiated cells showed positive GFAP immunoreactivity. Conclusion: NSCs grows well in serum-free culture and can differentiate into neurons and astrocytes in serum-containing medium. The second part of the study on the differentiation of neural stem cells into hair-like cells by co-culture of neural stem cells with the supernatant of basal membrane of neonatal rats in vitro Aim: to investigate the differentiation of neural stem cells from embryonic rat neural stem cells into hair-like cells in vitro. Methods: NSCs were isolated from the brains of SD embryos and cultured in serum-free medium. The supernatant of the supernatant was extracted from the basement membrane of the neonatal rat and cultured with NSCs in vitro. After 14-21 days, the hair cell markers myosin VIIa and calretinin were detected by immunofluorescence and immunohistochemistry, and the differentiation ratio was calculated. Results: about 15.4% of the cells differentiated by NSCs showed myosin VIIa immunofluorescence positive and 21. 7% were calretinin positive. Conclusion: the supernatant of basal membrane of neonatal rats can induce the differentiation of NSCs into hair-like cells in vitro. The third part of the study on differentiation of neural stem cells into hair-like cells after co-culture with neonatal rat basement membrane Aim: to investigate the differentiation of neural stem cells into hair-like cells in vitro. Methods: NSCs were isolated from the brains of SD embryos and cultured in serum-free medium. The basement membrane of neonatal rats was cultured with NSCs within 3 days. After 14-21 days, the hair cell markers myosin VIIa and calretinin were detected by immunofluorescence and immunohistochemistry, and the differentiation ratio was calculated. Results: about 17.1% of the cells differentiated by NSCs showed myosin VIIa immunofluorescence positive and 27. 4% were calretinin positive. Conclusion: the basal membrane and NSCs can induce the neonatal rat to differentiate towards the hair cells.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329;R764

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