低氧对人骨髓间充质干细胞向神经细胞分化的影响

目的 探讨低氧对人骨髓间充质干细胞(hBMSCs)向神经细胞分化的影响.方法 将第2代hBM-SCs进行传代、纯化,取第4代hBMSCs进行流式细胞仪检测鉴定;以含1％ FBS的α-MEM为基础诱导培养基,根据处理条件不同分为4组:常氧对照组、低氧对照组(3％氧浓度)、常氧诱导组[添加20 ng/mL表皮生长因子(E GF)及20 ng/mL碱性成纤维细胞生长因子(bFGF)]、低氧诱导组(3％氧浓度下添加20 ng/mL EGF及20 ng/mL bFGF).连续诱导3d后,采用RT-PCR及Western blot技术检测神经元特异性烯醇化酶(NSE)、微管相关蛋白2(MAP-2)及胶质纤维酸性蛋白(GFAP)在基因及蛋白水平表达变化.结果 第4代hBMSCs流式鉴定结果示:骨髓基质标志CD105、CD90、CD29表达分别为95.8％、98.2％、95.1％,而造血细胞标志CD19表达为0.2％;与常氧对照组比较,低氧对照组NSE、MAP-2、GFAP在基因及蛋白水平表达均无明显改变(P＞0.05),而常氧诱导组及低氧诱导组相应表达均升高(P＜0.05),且低氧诱导组表达高于常氧诱导组(P＜0.05).结论 低氧促进EGF联合bFGF对hBMSCs向神经元细胞及神经胶质细胞分化的诱导作用.

Effects of hypoxia on the differentiation of human bone marrow mesenchymal stem cells into neural cells

Objective To investigate the effect of hypoxia on the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) into neural cells.Methods The hMSCs (Passage 2) were purchased from Cyagen Biosciences Inc and identified by flow cytometry at Passage 4.The α-MEM medium containing 1％ FBS was used as the basal inducing medium.According to culture conditioning,they were divided into 4 groups,normoxia group,hypoxia group (3％ O2),normoxia-induced group (adding 20 ng/mL EGF and 20 ng/mL bFGF),and hypoxia-induced group (adding 20 ng/mL EGF and 20 ng/mL bFGF under the condition of 3％ O2).RT-PCR and Western blot were used to assess the mRNA and protein levels of the neuron markers,including NSE and MAP-2,and GFAP for glial marker after continuous inducing of 3 days.Results The identification results of flow cytometry showed that the expression of bonemarrow stroma marker CD105,CD90 and CD29 were 95.8％,98.2％ and 95.1％,respectively;while the expression of CD19,as the marker of hematopoietic cell,was 0.2％.Compared to normoxia group,the mRNA and protein levels of NSE,MAP-2 and GFAP were similar to hypoxic-group,while those were significantly increased both in normoxia-induced group and hypoxia-induced group (P ＜ 0.05);between which,they were significantly higher in hypoxia-induced group (P ＜ 0.05).Conclusion Hypoxia promotes the induction of EGF combined with bFGF on differentiation of hBM-SCs into neural cells and neurogliocytes.