EPO促进胚胎神经干细胞向神经元分化的形态学研究

Study of morphology about the promotion of erythropoietin to the differentiation of the cultured NSCs from brain cortex in vitro

目的探讨促红细胞生成素(erythropoietin,EPO)促进神经干细胞(neural stem cells,NSCs)分化的形态学表现,为细胞移植治疗脑部疾病和脑损伤提供实验依据。方法取大鼠14d胚胎脑皮质先增殖培养,后贴壁分化培养。适时镜下观察,免疫细胞化学染色,用nestin鉴定NSCs,GFAP鉴定神经胶质细胞、MAP2鉴定神经元。选取第3代NSCs,向培养基中分别添加不同剂量的EPO,浓度分别为0.5u/ml,5u/ml,50u/ml,500u/ml,并设不添加EPO的对照组,MAP2免疫荧光共聚焦显微镜观察NSCs向神经元方向的分化。结果1)鼠胚脑皮质在无血清悬浮培养形成大量神经球,并可传代,球内细胞均表达nestin。分化培养,可检测出MAP2或GFAP阳性细胞。2)EPO≥5u/ml各组分化培养,表达MAP2阳性细胞显著升高(P<0.05)。结论大鼠胚胎脑皮质体外培养可获得NSCs;适当浓度EPO可提高NSCs向神经元的分化。

Objective Methods Results Conclusion To investigate the promotion of EPO on the differenti-ation of the neural stem cells （NSCs） in vitro from cerebral cortex of pregnant SD rats .The brain cortex were iso-lated and cultured in serum-free suspension ,and then in differentiating suspension .Nestin was used to detect the NSCs .Glia fibrillary acid protein （GFAP） and microtubule-associated protein 2 （MAP2 ） were used to detect the dif-ferentiation of NSCs by immunocytochemistry .After obtained the third passage （P3 ） of NSCs ,EPO of different concentration （0.5 ,5 ,50 ,500u/ml） was added to the medium .And there was a control group without EPO .MAP2 was used to detect the neuron after the differentiation by immunocytochemistry .The results showed that the sepa-rated cells from embryonic brain cortex formed neurospheres ,in which nestin-positive cells were detected .GFAP and MAP2-positive cells were detected after differentiation culture .MAP-2-positive cells of the EPO supplementa-tion groups （＞5U/ml） showed an significant increase in the immunochemistry dying test compared with the con-trol group .The results indicate that NSCs can be obtained from the separated cerebral cortex of embryonic rats .Ap-propriate concentration of EPO can increase the rate of the neuron after the differentiation the NSCs in vitro .