EPO对鼠胚脑皮质神经干细胞抗凋亡作用的研究

Study of anti-apoptotic effect of erythropoietin on the cultured NSCs from brain cortex of embryonic rats in vitro

目的探讨促红细胞生成素(erythropoietin,EPO)对体外培养神经干细胞(neural stem cells,NSCs)凋亡的影响,为NSCs移植治疗脑损伤和脑部疾病提供实验依据。方法孕14 d(El4)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,用免疫细胞化学染色以Nestin蛋白作为鉴定NSCs的标记物,以MAP2、GFAP蛋白检测NSCs的分化。取第3代(P3)NSCs向悬浮培养基中分别添加不同剂量的EPO,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,通过Caspase-3蛋白检测NSCs的凋亡。结果 1)提取和分离E14d SD大鼠的胚脑皮质,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。2)加入≥5U/ml EPO,神经球内Caspase-3的表达显著下降(P<0.01)。结论 1)大鼠胚胎脑皮质体外培养可得到大量增殖的NSCs并能分化为神经元和神经胶质细胞,为NSCs的研究提供了适宜的细胞培养模型。2)加入≥5U/ml EPO可降低体外培养的鼠胚脑皮质NSCs的凋亡率。

Objective To explore the effects of EPO on the apoptosis of the neural stem cells （NSCs） in vitro from brain cortex of embryonic Sprague-Dawley rats. Methods The NSCs were isolated and cultured in serumfree suspension, then cultured by differentiating suspension. Nestin was used to detect the NSCs. Microtubule-associated protein 2（MAP2） and glial fibrillary acid protein （GFAP） was used to detect the differentiation of NSCs by immunofluorescent cytochemistry. EPO was added to the serum-free suspension. According to the final EPO concentration, the cells were divided into 4 experimental groups, they were 0.5 ; 5,50,500U/ml, in addition the control group was without EPO. Caspase-3 was measured to analyze the apoptosis of NSCs by immunofluorescent cytochemistry. Results The results showed that separated cells from brain cortex could proliferate and subculture in vitro. Nestin-positive cells were detected in neural stern spheres. MAP2- and GFAP-positive cells were detected in serum with 10 % FBS. Caspase-3-positive NSCs in EPO supplementation groups（〉5U/ml） were decreased in neural stem spheres compared with the control group. Conclusion Our results indicate that the separated cells from brain cortex of embryonic rats are multipotent and have the ability of self-renew and differentiation, which suggest that it may be a good cell culture model for the study of NSCs. The present results suggest that EPO can decrease the apoptosis of the NSCs in vitro.