子宫内电穿孔法调控鼠胚大脑皮质神经干细胞基因表达体系的建立

Establishment of a system for regulating the gene expression of embryonic mouse cerebral cortex neural stem cells by in utero electroporation

摘要:

目的 利用子宫内电穿孔（in utero electroporation，IUE）技术，建立小鼠胚胎阶段调控大脑皮质神经干细胞基因表达体系。 方法 向孕14.5 d鼠胚脑室内注入pCIG质粒，电转至大脑皮质神经干细胞（neural stem cell，NSC）。取孕16.5 d或孕17.5 d鼠胚脑组织制作冰冻切片，采用免疫荧光染色观察NSC的增殖、凋亡、分裂、定向分化及迁移和成熟。 结果 可在孕16.5 d观察到NSC向中间前体神经元分化、NSC增殖与凋亡及放射状胶质细胞放射轴形态结构发育的情况；可在孕17.5 d观察到NSC向大脑皮质Ⅴ~Ⅵ层神经元分化、NSC向外侧大脑皮质迁移、迁移神经元树突发育及神经元成熟的情况。 结论 采用IUE技术可成功建立调控小鼠胚胎大脑皮质NSC基因表达体系，这有利于深入开展大脑皮质NSC的增殖、凋亡、分裂、定向分化、迁移和成熟等神经发育相关研究。 ［中国当代儿科杂志，2022，24（9）：1061-1067］

Abstract:

Objective To establish a system for regulating the gene expression of embryonic mouse cerebral cortex neural stem cells (NSCs) using in utero electroporation (IUE). Methods At embryonic day 14.5, the mouse cerebral cortex NSCs were electro-transfected with the pCIG plasmid injected into the ventricle of the mouse embryo. At embryonic day 16.5 or day 17.5, embryonic mouse brain tissues were collected to prepare frozen sections. Immunofluorescence staining was used to observe the proliferation, apoptosis, division, directional differentiation, migration, and maturation of NSCs. Results The differentiation of NSCs into intermediate progenitors, the proliferation and apoptosis of NSCs, and the morphological development of radial axis of radial glial cells were observed at embryonic day 16.5. The differentiation of NSCs into neurons in layers V-VI of the cerebral cortex, the migration of NSCs to the lateral cerebral cortex, the development of dendrites of migrating neurons, and the maturation of neurons were observed at embryonic day 17.5. Conclusions The system for regulating the gene expression of embryonic mouse cerebral cortex NSCs can be established using IUE, which is useful for the study of neural development related to the proliferation, apoptosis, division, directional differentiation, migration and maturation of NSCs in the cerebral cortex. Citation:Chinese Journal of Contemporary Pediatrics, 2022, 24(9): 1061-1067