大鼠神经干细胞的体外分离培养及分化研究

发布时间：2018-09-01 20:06

【摘要】：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(P0.05),GFAP的表达逐渐减少(P0.05)。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。
[Abstract]:Objective to establish a method for isolation, differentiation, identification and long-term culture of neural stem cells (NSCs) from neonatal SD rats, and to explore the effect of serum concentration on NSCs differentiation. Methods the brain tissue of newborn SD rats was taken and NSCs. was cultured by serum-free culture. NSCs was identified by immunofluorescence technique and its nestin (Nestin) expression was detected. The differentiation of NSCs was induced by DMEM/F-12 medium containing 10% fetal bovine serum (FBS). The differentiated cells were identified by immuno fluorescence staining with neuron specific microtubule-associated protein 2 (MAP-2) antibody and glial cell-specific glial fibrillary acidic protein (GFAP) antibody. The expression of MAP-2 and GFAP in different concentration of FBS was detected by Western blot method. Results A large number of undifferentiated and suspended NSCs spheres were obtained and differentiated into neurons and glial cells. With the increase of serum concentration, the expression of MAP-2 increased (P0.05) and the expression of GFAP decreased (P0.05). Conclusion using serum-free culture technique, neonatal rat NSCs, with proliferation, self-renewal and multipotential differentiation can be successfully cultured in vitro and further differentiated into neurons and glial cells under the conditions of different FBS concentration (22% and 10%). The conditioned medium containing low concentration of serum could promote the differentiation of NSCs into neurons, while high concentration of serum could promote the differentiation of NSCs into glial cells.
【作者单位】：安徽医科大学第一附属医院骨科;安徽医科大学基础医学院组织学与胚胎学教研室组织工程干细胞研究所;
【分类号】：R329

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