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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 culture ventral midbrain neural stem cells (NSCs) from rat embryos in vitro and observe their growth and differentiation characteristics. Methods: The SD rats of 12.5 ~ 14.5 d were enrolled and the ventral midbrain tissues were obtained by mechanical separation. The cells were cut into pieces and single-cell suspension. The cells were seeded at a density of 2 × 10 ~ 5 cells / ml Serum-free NSCs culture medium by neurospheres. The neurospheres were cultured for 5 to 7 days and subcultured according to the original density. The third generation neurospheres were differentiated with 10% fetal bovine serum (FBS) differentiation medium and differentiated for 7 days. Immunocytochemistry Identification. Results: The neurospheres were positive for nestin. The differentiated cells were positive for βIII-Tubulin and glial fibrillary acidic protein (GFAP). CONCLUSION: The ventral neural stem cells from the ventral midbrain of rat embryos cultured by neurospheres can proliferate and differentiate into neurons and glial cells.