胚胎期Wnt3a基因特异性敲降对小鼠大脑神经发生的影响  

Effect of the specific knockdown of Wnt3a in embryonic stage on neurogenesis in mouse brain

作　　者：赵程 焦建伟 ZHAO Cheng;JIAO Jianwei(State Key Disciplines:Physiology(Incubation),Department of Physiology,Qingdao University,Qingdao 266071,China)

机构地区：[1]青岛大学国家生理学重点(培育)学科,山东青岛266071 [2]中国科学院动物研究所干细胞与生殖学国家重点实验室

出　　处：《青岛大学学报（医学版）》2024年第3期355-358,共4页Journal of Qingdao University(Medical Sciences)

基　　金：国家自然科学基金重点项目(32230040)。

摘　　要：目的 研究胚胎期第13天(E13)特异性敲降Wnt3a基因对小鼠大脑神经发生的影响。方法 利用实时荧光定量PCR(RT-qPCR)验证Wnt3a在脑皮质神经干细胞中的敲降率。通过子宫内电穿孔实验在E13时将Wnt3a-shRNA质粒电转到胎鼠大脑皮质中,从而下调Wnt3a在脑皮质神经干细胞中的表达。在E16时收取小鼠脑组织,进行免疫荧光染色检测,观察下调Wnt3a后小鼠脑组织中神经元发育进程的改变。结果 RT-qPCR结果显示,神经干细胞中Wnt3a敲降后其mRNA表达水平降低54%,与对照组相比差异具有统计学意义(t=11.260,P<0.001)。免疫荧光染色结果显示,Wnt3a下调导致大脑皮质的皮质板(CP)中的绿色荧光蛋白(GFP)阳性细胞比例降低,大脑中间带(IZ)的GFP阳性细胞比例上升,差异均具有统计学意义(t=4.400、4.482,P<0.01)。结论 E13时特异性下调小鼠脑中Wnt3a基因导致GFP荧光分布异常,神经发生过程受到干扰。Objective To investigate the effect ofspecific knockdown of the Wnt3a gene at embryonic day 13(E13)on brain neurogenesis in mice.Methods RT-qPCR was used to verify the knockdown rate of Wnt3a in neural stem cells in the cerebral cortex,and intrauterine electroporation was used to transfect Wnt3a-shRNA plasmid into the cerebral cortex of fetal mice at E13 to downregulate the expression of Wnt3a in neural stem cells in the cerebral cortex.The mouse brain tissue samples were collected at embryonic day 16(E16),and immunofluorescent staining was performed to observe the changes in neuronal development after the downregulation of Wnt3a.Results RT-qPCR showed that the mRNA expression level of Wnt3a in neural stem cells was reduced by 54%after knockdown,with a significant difference compared with the control group(t=11.260,P<0.001).Immunofluorescent staining showed that the downregulation of Wnt3a led to a reductionin the proportion of green fluorescent protein(GFP)-positive cells in the cortical plateof the cerebral cortexand an increase in the proportion of GFP-positive cells in the intermediate zone(IZ)(t=4.400,4.482;P<0.01).Conclusion The specific downregulation of the Wnt3a gene in the brain of mice at E13 can lead to an aberrant distribution of GFP fluorescence,thereby disrupting the process of neurogenesis.

关 键 词：Wnt3A蛋白质 小鼠 基因敲除 神经发生 大脑皮质 胚胎发育 电穿孔 

分 类 号：R977.6[医药卫生—药品] R329.21[医药卫生—药学]