NT-3高表达促进神经干细胞向胆碱能神经元分化  

Neurotrophin 3 gene overexpression promotes cholinergic differentiation in neural stem cells

作　　者：闫宇辉[1] 李少恒[1] 孔亮[1] 姚璎珈 教亚男 陶震宇 宋捷[1] 杨静娴[1] 

机构地区：[1]辽宁中医药大学药学院药理学教研室,辽宁大连116600

出　　处：《中国药理学通报》2016年第5期631-637,共7页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金面上项目(No 81173580)

摘　　要：目的体外研究神经营养因子3(neurotrophin-3,NT-3)基因转染神经干细胞(neural stem cells,NSCs)后对其向胆碱能神经元分化的影响,并探讨其机制。方法体外分离培养新生小鼠脑源NSCs,免疫荧光细胞化学法对其进行鉴定;将NSCs分为NSCs组(不作任何处理的NSCs)、GFP-NSCs组(转染GFP的NSCs)、NT-3-NSCs组(转染NT-3的NSCs),免疫荧光细胞化学法和ELISA法检测各组NSCs中NT-3的表达;免疫荧光细胞化学法和RT-PCR法检测各组NSCs向胆碱能神经元分化的能力;乙酰胆碱检测试剂盒检测乙酰胆碱分泌情况;RT-PCR法检测Notch信号通路相关靶基因Hes1、Mash 1和Neurogenin 1(Ngn1)表达情况。结果免疫荧光细胞化学法结果显示,NSCs表达其特异性标志蛋白Nestin和Sox2,与NSCs组和GFP-NSCs组相比,NT-3-NSCs组能够分化为更多的胆碱能神经元(P<0.01),分化的胆碱能神经元可分泌乙酰胆碱(P<0.01),且能够减少Notch通路靶基因Hes 1 mRNA的表达,增加Mash1、Ngn 1 mRNA的表达(P<0.05)。结论 NT-3高表达可促进NSCs分化为更多的胆碱能神经元,其机制可能与抑制Notch信号通路有关。Aim To investigate the effects of neurotro-phin-3 ( NT-3 ) gene overexpression on the differentia-tion into cholinergic neuron of neural stem cells ( NSCs) in vitro and its underlying mechanism. Meth-ods Brain-derived NSCs from newborn mice were iso-lated and cultured in vitro and determined by immuno-fluorescence. The NSCs were divided into three groups: NSCs, GFP-NSCs and NT-3-NSCs groups. The expression of NT-3 was detected by immunofluo-rescence and ELISA. Then, the ability of NSCs on dif-ferentiation into cholinergic neuron was detected by im-munofluorescence and RT-PCR, and the Acetylcholine Assay Kit was used for acetylcholine ( ACh) , and the expression of Hes1 , Mash1 and Ngn1 mRNA was de-termined by RT-PCR. Results The neurosphere dis-played Nestin and Sox 2-postive by immunofluores-cence, suggesting that the cultured cells were NSCs. The proportion of ChAT immunopositive cells was sig-nificantly higher in the NT-3-NSCs group than that in the other two groups ( P <0. 01 ) . Ach secretion in NT-3-NSCs was significantly elevated compared with the other two groups ( P <0. 01 ) . NSCs transfected with NT-3 increased the levels of Mash1 and Ngn1 mR-NA, and decreased the level of Hes1 mRNA ( P <0. 05 ) . Conclusion NT-3 can significantly promote the in vitro differentiation of NSCs into cholinergic neu-rons via probablly inhibiting Notch signaling pathway.

关 键 词：神经干细胞 阿尔茨海默病 神经营养因子3 胆碱能神经元 分化 NOTCH信号通路 

分 类 号：R-332[医药卫生] R322.8