【摘要】 目的:观察脑因子4(Brn4)对神经干细胞(NSCs)向神经元分化的诱导作用,探讨Brn4促进新生神经元成熟和维持细胞存活的分子机制。方法:应用基因重组技术,构建pLV5-Brn4-GFP慢病毒表达载体,感染NSCs后使外源性Brn4基因稳定高表达。NSCs诱导分化后,应用细胞免疫荧光法检测NSCs分化为微管相关蛋白-2(MAP-2)阳性神经元的情况,应用TUNEL法检测分化细胞的凋亡情况。结果:重组病毒感染NSCs后,Brn4蛋白表达较对照组增高约5倍,同时分化细胞中胶质细胞源性神经营养因子(GDNF)及其受体GFRα-1和Ret蛋白的相对表达量均明显高于对照组(P﹤0.01)。NSCs分化2周后,Brn4过表达组MAP-2阳性神经元的分化率(65.71±5.18%)明显高于对照组(12.57±1.77%)(P﹤0.01),且细胞胞体较大,突起较丰富。而凋亡细胞的百分率(2.73±0.44%)明显低于对照组(8.39±0.95%,P﹤0.01)。结论:Brn4基因过表达可诱导NSCs向神经元分化,同时通过GDNF信号通路促进新生神经元的发育成熟,并通过抑制细胞凋亡提高细胞存活率。更多还原

【Abstract】 Objective: To observe the induction effect of Brn4 on the neuronal differentiation of neural stem cells( NSCs),and to investigate the molecular mechanism of Brn4 on promoting the maturation of new-born neurons and sustaining cell survival. Methods: The lentiviral expression vector pLV5-Brn4-GFP was constructed by gene recombination technology and infected NSCs to induce the stable and strong expression of exogenous Brn4 gene. The expressions of microtubule associated proteins-2( MAP-2) were detected by immunofluorescence labeling after the differentiation of NSCs,and the cell apoptosis was detected by TUNEL method. Results: After infection of the recombinant virus,the relative expression of Brn4 protein in NSCs was increased about 5 times compared with the control group. At the same time,the expression of GDNF and its receprots GFRα-1 and Ret proteins were markedly increased as compared with the control group( P ﹤ 0. 01). In the Brn4 group,the differentiation rate of MAP-2 positive neurons( 65. 71 ± 5. 18%) was obviously higher than control group( 12. 57 ± 1. 77%,P ﹤ 0. 01),and the cell bodies were larger,the neurites were richer.While the the percentage of apoptotic cells( 2. 73 ± 0. 44%) was significantly lower than control group( 8. 39 ± 0. 95%,P ﹤ 0. 01). Conclusion: The overexpression of Brn4 gene could induce NSCs to differentiate into neurons,and promotethe maturation of new-born neurons,improve the survival rate of cells through inhibiting cell apoptosis.更多还原