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目的检测体外培养SD大鼠胚脑皮质神经干细胞(neural stem cells,NSCs)增殖和分化的生物学特性,为NSCs研究提供适宜的细胞模型;探讨促红细胞生成素(erythropoietin,EPO)对NSCs增殖的影响,为NSCs的相关研究提供实验依据。方法本研究对孕14d(E14)SD大鼠取鼠胚脑皮质悬浮培养、贴壁诱导分化。采用光电镜观察,以nestin免疫荧光染色鉴定NSCs,微管相关蛋白2(microtubule associated protein2,MAP2)和神经胶质原纤维酸性蛋白(glia fibrillary acid protein,GFAP)检测NSCs分化。取第三代(P3)NSCs向悬浮培养基中添加不同剂量的EPO,通过四甲基偶氮唑蓝(MTT)检测法检测NSCs的增殖情况。结果分离E14dSD大鼠胚脑皮质,在添加B27、bFGF、EGF的无血清培养基中培养,可形成大量悬浮的神经球并可进行体外扩增传代,神经球内的细胞均呈Nestin阳性、BrdU阳性。在添加10%胎牛血清的培养基中,神经干细胞可自然分化为神经元和神经胶质细胞。与对照组对比,加入≥5U/mlEPO后MTT检测NSCsOD值明显增高。结论 SD大鼠胚脑皮质体外培养可得到大量增殖的神经干细胞并能分化为神经元和神经胶质细胞,EPO可促进体外NSCs的增殖。
Objective To detect the biological characteristics of proliferation and differentiation of neural stem cells (NSCs) cultured in vitro of SD rats and to provide suitable cell models for the study of NSCs. To investigate the effect of erythropoietin (EPO) on the proliferation of NSCs Influence, provide the experimental basis for the related research of NSCs. Methods In this study, embryonic 14d (E14) SD rats were cultured in mouse cerebral cortex suspension and adherently differentiated. The NSCs were identified by nestin immunofluorescence staining and microtubule associated protein 2 (MAP2) and glial fibrillary acid protein (GFAP) were detected by light microscopy. The third generation (P3) NSCs were added to the suspension medium with different doses of EPO, and the proliferation of NSCs was detected by MTT assay. Results The embryonic cortex of E14dSD rats was isolated and cultured in serum-free medium supplemented with B27, bFGF and EGF to form a large number of neurospheres suspended and proliferated and passaged in vitro. The cells in the neurospheres were positive for Nestin and BrdU Positive. Neural stem cells naturally differentiate into neurons and glial cells in medium supplemented with 10% fetal bovine serum. Compared with the control group, NSCsOD value increased significantly by MTT assay after adding 5U / ml EPO. Conclusion The cultured embryonic cortex of SD rats can produce large numbers of proliferating neural stem cells and differentiate into neurons and glial cells. EPO can promote the proliferation of NSCs in vitro.