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目的 构建Gabra3基因敲除细胞模型，探讨敲除Gabra3基因后小鼠神经元细胞在转录、蛋白水平的变化，研究Gabra3基因敲除后睡眠深度增加的机制。方法 构建多种sgRNA序列，使用CRISPR/Cas9系统敲除鼠源GT1-7神经元细胞系中的Gabra3基因，通过基因测序检测敲除效率，使用TA克隆、蛋白免疫印迹实验验证敲除结果，细胞增殖实验验证敲除表型，并提取总RNA进行转录组测序，提取总蛋白进行蛋白质测序。结合多种生物信息学分析方法，评估Gabra3基因敲除对GT1-7神经元细胞功能的影响。结果 Gabra3基因敲除后，皮质醇和醛固酮的合成与分泌、昼夜节律等通路显著富集，并筛选出BMP2、GLI2、DLL1三个关键基因，蛋白组结果显示Gabra3基因敲除后产生了广泛的扰动。结论 Gabra3基因敲除可能通过调控激素分泌相关基因表达，提高睡眠深度。
Abstract：Objective To construct a Gabra3 gene knockout cell model and explore transcriptomic and proteomic alterations in murine neuronal cells, in order to investigate the molecular mechanisms underlying the increased depth of slow-wave sleep observed following Gabra3 deletion. Methods Multiple sgRNA sequences were designed, and the CRISPR/Cas9 system was used to knock out the Gabra3 gene in the murine GT1-7 neuronal cell line. Gene sequencing was performed to assess knockout efficiency, and TA cloning was used to validate the knockout results. Protein immunoblotting experiments were conducted to confirm the knockout, while cell proliferation assays were used to validate the knockout phenotype. Total RNA and protein were extracted for transcriptomic and proteomic sequencing, respectively. A range of bioinformatics analyses was conducted to assess the functional consequences of Gabra3 knockout in GT1-7 neuronal cells. Results Following Gabra3 gene knockout, pathways related to cortisol and aldosterone synthesis and secretion, as well as circadian rhythm, were significantly enriched. Three key genes, BMP2, GLI2, and DLL1, were identified. Proteomic profiling revealed widespread disturbances in protein expression following Gabra3 knockout. Conclusion Gabra3 gene knockout may increase slow-wave sleep depth by modulating the expression of hormone secretion-related genes and altering circadian regulatory pathways.
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基本信息:
DOI：10.16289/j.cnki.1002-0837.2025.03003
中图分类号:R856.74
引用信息:
[1]张力中,唐育杰,孙毅等.Gabra3基因敲除对小鼠神经元细胞睡眠功能影响的探究[J].航天医学与医学工程,2025,36(03):206-213.DOI:10.16289/j.cnki.1002-0837.2025.03003.
基金信息:
国家重点研发计划(2021YFC2701704); 军队后勤自主科研课题(2022HQZZ06); 山西省基础研究计划(202303021221132); 山西省科技创新人才团队专项资助(202304051001017)