低氧微环境促进嗅黏膜间充质干细胞向神经元分化及其相关机制  

Hypoxic condition promotes olfactory mucosa mesenchymal stem cells to differentiate into neurons and underlying mechanisms

作　　者：卓毅[1] 袁挺[1] 段答[1] 王磊[1] 葛丽特[1] 吴沛[1] 王昊[1] 卢明[1] 

机构地区：[1]解放军第163医院/湖南师范大学第二附属医院神经外科,长沙410003

出　　处：《中南大学学报（医学版）》2016年第12期1252-1259,共8页Journal of Central South University ：Medical Science

基　　金：国家自然科学基金(81371358);湖南省自然科学基金(14JJ2060);湖南省教育厅湖南省研究生创新性项目(CX2015B188)~~

摘　　要：目的:研究低氧微环境下利用嗅鞘细胞(olfactory ensheathing cells,OECs)上清液诱导嗅黏膜间充质干细胞(olfactory mucosa mesenchymal stem cells,OM-MSCs)更多地向神经元分化及其相关的机制。方法:分离培养OECs和OM-MSCs,应用流式细胞仪和免疫荧光方法分别进行鉴定;实验将OM-MSCs分为3%O_2+低氧诱导因子-1α(hypoxiainducible factor-1α,HIF-1α)抑制剂利非西呱(lifi ciguat,YC-1)+OECs上清诱导组(A组)、3%O_2+OECs上清诱导组(B组)及21%O_2+OECs上清诱导组(对照组)。采用免疫荧光检测分化后的神经元,采用定量聚合酶链反应(quantitative polymerase chain reaction,Q-PCR)和Western印迹分别检测HIF-1α的mRNA及βIII-微管蛋白(βIII-tubulin)、胶质纤维酸性蛋白(glial fibrillary acidic portein,GFAP)的表达,再用膜片钳检测分化后的神经元钾离子通道开放情况。结果:实验各组相比较,B组OM-MSCs诱导后免疫荧光显示βIII-tubulin表达最多,Q-PCR显示B组HIF-1α表达明显增高(均P<0.05);Western印迹表明B组中βIII-tubulin蛋白表达显著增多,而胶质细胞标志物GFAP表达明显降低(均P<0.05);且诱导分化后的神经元经膜片钳检测发现钾离子电流。结论:低氧微环境下的OM-MSCs经OECs上清液诱导能显著提高其向神经元分化的比例,并明显降低了神经胶质细胞的产生,且与细胞HIF-1α信号通路被激活有关。Methods： The OM-MSCs and OECs were isolated and cultured, and they were identified by flow cytometry and immunofluorescence. The OM-MSCs were divided into three groups： a 3%02＋ HIF-1α inhibitors （lificiguat： YC-1） ＋ OECs supernatant group （Group A）, a 3%O2 ＋ OECs supernatant group （Group B） and a 21%O2 ＋ OECs supernatant group （Control group）. The neurons, which were differentiated from OM-MSCs, were assessed by immunofluorescence test. The mRNA and protein expression of hypoxia-inducible factor-1α （HIF-1α）, βⅢ-tubulin and glial fibrillary acidic portein （GFAP） were detected by quantitative polymerase chain reaction （Q：PCR） and Western blot. The potassium channels were analyzed by patch damp. Results： The neurons differentiated from OM-MSCs expressed the most amount of [3III-tubulin, and the result of Q-PCR showed that HIF-la expression in the Group B was significantly higher than that in the other groups （all P〈0.05）. Western blot result showed that the βⅢ-tubulin protein expression was significantly higher and GFAP protein expression was obviously decreased in the Group B （both P〈0.05）. The patch damp test confirmed that the potassium channels in the neurons were activated. Conclusion： Hypoxic condition can significantly increase the neuronal differentiation of OM- MSCs by the OECs supematant and decrease the production of neuroglia cells, which is associated with the activation of HIF-1 signal pathway.

关 键 词：低氧微环境 低氧诱导因子-1Α 嗅黏膜间充质干细胞 嗅鞘细胞 神经元 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]