促红细胞生成素对神经干细胞缺氧性损伤保护作用的实验研究

PROTECTIVE EFFECT OF EPO ON HYPOXIA-IMPAIRED NSCs

目的观察外源性EPO对神经干细胞缺氧性损伤的保护作用,为缺氧缺血性脑损伤的治疗提供新思路。方法从孕11·5d(E11·5d)大鼠获得神经干细胞,经无血清培养基悬浮培养并传代,对所获细胞的自我增殖、自我更新及其多分化潜能进行检测。取传3代神经干细胞中添加不同剂量的EPO,在5%O2培养箱中培养120h。通过计数干细胞克隆形成率和MTT法检测神经干细胞的增殖情况。于含血清分化培养基中加入不同剂量的EPO,用NSE和GFAP免疫细胞化学染色观察神经干细胞的分化情况。采用AnnexinⅤ-FITC/PI染色,激光扫描共聚焦显微镜观察、检测细胞凋亡率。结果加入EPO后神经干细胞的克隆形成率和MTT检测的OD值显著增高,细胞凋亡率显著下降,NSE阳性细胞的比例明显升高,其作用随剂量增加而增大,50U/ml时作用最大。结论EPO对神经干细胞缺氧性损伤具有明显的保护作用,并可促进神经干细胞向神经元方向分化。EPO的这种作用随剂量增加而增大,50U/ml时达高峰。

Objective To observe the protection of exogenous EPO on NSCs impaired by hypoxia and to provide a new therapeutic method for hypoxie-ischemic brain injury. Methods The NSCs of E11. 5d were isolated, cultured and passaged. Different concentrations of EPO were added to cultures during proliferation of NSCs exposed to 5%O2 for 120h. Proliferation of NSCs was evaluated by MTT method and calculating clones. During the differentiation of NSCs with 10% FBS, different concentrations of EPO were added. The ratio of the differentiated neurons to glia cells was detected and counted by NSE and GFAP immunocytochemical staining. Apoptosis was measured by Annexin V-FITC/PI immunofluorescent staining and laser scanning cofocal microscopy. Results The EPO supplementation group displayed a significant increase of clone and OD value, but striking decrease in apoptosis during proliferation. NSE positive cells increased significantly in the EPO supplementation group than in the control group during differentiation. When the concentration of EPO reached 50U/ml, the dose-dependent protection of EPO was maximized. Conclusion EPO can evidently protect NSCs against hypoxia, and at the same time, induce NSCs to differentiate into neuron. The dose-dependent protection effect of EPO is maximal at the concentration of 50 U/ml.