大鼠神经干细胞的体外分离培养及分化研究

Isolation,cultivation in vitro and differentiation of rat neural stem cells

目的建立新生SD大鼠神经干细胞(NSCs)的分离培养、体外分化、鉴定及体外长期培养的方法,并探讨血清浓度对NSCs分化的影响。方法取新生SD大鼠大脑组织,用无血清培养技术培养NSCs。采用免疫荧光技术对NSCs进行鉴定,检测其巢蛋白(Nestin)表达。用含有不同胎牛血清(FBS)浓度(2%、5%、10%)的DMEM/F-12培养基诱导NSCs分化,分别用神经元特异性微管相关蛋白2(MAP-2)抗体、神经胶质细胞特异性胶质纤维酸性蛋白(GFAP)抗体行免疫荧光染色鉴定分化细胞。通过Western blot法检测不同FBS浓度的分化条件下MAP-2与GFAP的表达情况。结果获得大量未分化且悬浮生长的NSCs球,并能分化为神经元和神经胶质细胞。随着血清浓度的增加,各组MAP-2的表达逐渐增加(P<0.05),GFAP的表达逐渐减少(P<0.05)。结论应用无血清培养技术可成功在体外培养出具有增殖、自我更新及多潜能分化能力的新生大鼠NSCs,可于含有不同FBS浓度(2%、5%、10%)的分化条件下进一步分化为神经元及神经胶质细胞,含低浓度血清的条件培养基促进NSCs向神经元方向分化,而高浓度血清则有利于NSCs向神经胶质细胞方向分化。

Objective To establish the isolation, differentiation, identification and long-term cultivation in vitro of the neural stem cells （NSCs） from neonatal SD rats, and to investigate the effect of the serum concentration on the differentiation of NSCs. Methods Brain tissue was isolated from neonatal SD rats and NSCs were cultured in serum free medium. To identify NSCs and detect Nestin expression in NSCs by immunofluorescence. In the condition of DMEM/F-12 containing different concentrations （2%, 5%, 10% ） of FBS to induct the differentiation of NSCs, to identify the differentiated cells with the antibodies of neuron specific microtubule-associated protein 2 （MAP-2） and glial cell specific glial fibrillary acidic protein （GFAP） by immunofluorescence. In the differentiation conditions containing different concentrations of FBS, to detect the expression of MAP-2 and GFAP by Western blot. Results A mass of undifferentiated neurospheres were obtained and cultured in suspension, those NSCs could differentiate into neurons and astrocytes. With increasing serum concentrations, the expression of MAP-2 in each group gradual- ly increased （P 〈 0.05 ）, and the expression of GFAP gradually decreased （ P 〈 0. 05 ）. Conclusion We success- fully obtain the fetal rat NSCs in vitro, which have the capacities of proliferation, self-renew and pluripotentiality with the application of serum free cultivation. In the differentiation conditions containing different concentrations （2% , 5% , 10% ） of FBS, NSCs differentiate into neurons and glial cells, the conditioned medium containing low concentration of serum promote the differentiation of NSCs into neurons, the high concentration of serum is condu- cive to the differentiation of NSCs into neural glial cells.