小鼠原代海马神经元培养方法优化及HT22-GRK2^(-/-)细胞构建  

Optimization of culture method of mouse primary hippocampal neurons and construction of HT22-GRK2^(-/-)cells

作　　者：郭梦慧 薛娜娜 袁溪 孟浅 魏伟[1] Guo Menghui;Xue Nana;Yuan Xi;Meng Qian;Wei Wei(Institute of Clinical Pharmacology,Anhui Medical University,Key Laboratory of Anti-inflammatory and Immune Medicine,Ministry of Education,Anhui Collaborative Innovation Center of Anti-inflammatory and Immuno Drugs,Hefei 230032)

机构地区：[1]安徽医科大学临床药理研究所,抗炎免疫药物教育部重点实验室,安徽省抗炎免疫药物协同创新中心,合肥230032

出　　处：《安徽医科大学学报》2023年第4期589-596,共8页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:82101311、81973332)。

摘　　要：目的 探究并优化新生小鼠海马神经元体外原代培养方法;构建小鼠海马神经元HT22细胞中G蛋白偶联受体激酶2(GRK2)基因敲除细胞株(HT22-GRK2^(-/-))。方法 为优化小鼠原代海马神经元的培养,取新生1~2 d C57BL6/J小鼠海马组织,经胰酶消化后吹打形成细胞悬液,于Neurobasal-A培养基中加细胞培养添加剂维持细胞生长。利用CRSIPR/Cas9基因编辑技术构建HT22-GRK2^(-/-)细胞株,并通过免疫荧光染色和Western blot检测GRK2敲除效率。结果 以3×10^(7)/孔密度接种的新生小鼠原代海马神经元以无血清培养方式接种于6孔板中,可获得纯度高、活性好的小鼠原代海马神经元细胞;HT22-GRK2^(-/-)细胞株构建成功。结论 成功建立并优化小鼠海马神经元体外原代培养方法,利用CRSIPR/Cas9基因编辑技术成功构建HT22-GRK2^(-/-)细胞株。Objective To explore and optimize the primary culture method of neonatal mouse hippocampal neurons in vitro.To construct a G-protein-coupled receptor kinase 2(GRK2)knockout HT22 cell line.Methods Hippocampal tissue of C57BL6/J mice on day 1-2 was taken,digested with trypsin and pipetted to form a cell suspension,and supplement was added to Neurobasal-A medium to maintain cell growth.CRSIPR/Cas9 gene editing technique was used to construct HT22-GRK2^(-/-) cell line,and the knockout efficiency of GRK2 was detected by immunofluorescence staining and Western blot.Results Primary hippocampal neurons of newborn mice were put into six-well plates with 3×10^(7)/well using a serum-free culture method,which could get a high purity and good activity;HT22-GRK2^(-/-) cell line was constructed successfully.Conclusion The primary culture method of mouse hippocampal neurons was successfully established and optimized,and HT22-GRK2^(-/-) cell line was successfully constructed by CRSIPR/Cas9 gene editing technique.

关 键 词：海马神经元 原代培养 HT22 GRK2 CRSIPR/Cas9 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]