促红细胞生成素促进体外鼠胚脑皮质神经干细胞增殖

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。

The proliferation of the cultured neural stem cells of cortex promoted by erythropoietin in SD rats

Objective The proliferation and differentiation of neural stem cells （NSCs） of cortex were studied in embryonic Sprague-Dawley rats in vitro, and the effect of erythropoietin （EPO）on the proliferation of the NSCs was explored. Methods The NSCs were isolated and cultured in serum-free suspension, then cultured by differentiating suspension and identified by nestin immunohistochemistry. The self-renewal of NSCs was detected using light microscope and electron microscope. Microtubule-associated protein 2（MAP2）and glia fibrillary acid protein（GFAP） were used to detect the differentiation of NSCs by immunofluoresocytochemistry. 0.5, 5, 50 and 500u/ml concentrations of EPO were added respectively to NSCs serum-free suspension to determined the proliferaton of NSCs by light microscope and MTT test. Results The cells of cortex in embryonic rats were isolated and cultured in serum-free suspension. Nestin-positive and BrdU-positive cells were detected in neural sphere, MAP2 and GFAP positive cells were found in serum with 10% FBS. The EPO supplementation groups displayed a significant increase of NSCs and OD values compared with the control group. Conclusion EPO can promote the proliferation of the NSCs in vitro with a concentration dependent manner.