一种改良原代海马神经元糖氧剥夺/复氧模型的建立  

Establishment of an improved model of oxygen glucose deprivation/reoxygenation of primary hippocampal neurons

作　　者：史晋朝 马娜 张倩[1] 陈静[1] 钟锦 周阳 李建国[1] SHI Jinchao;MA Na;ZHANG Qian;CHEN Jing;ZHONG Jin;ZHOU Yang;LI Jianguo(Key Laboratory of Cellular Physiology,Ministry of Education,Shanxi Medical University,Taiyuan 030001,Shanxi Province,China)

机构地区：[1]山西医科大学细胞生理学教育部重点实验室,山西太原030001

出　　处：《中国生物制品学杂志》2024年第4期471-474,480,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81671231);山西省回国留学人员基金(2017-056);山西省“1331工程”重点学科建设计划(XK201708);山西省基础研究计划面上项目(20210302123305)。

摘　　要：目的 建立一种改良的原代海马神经元糖氧剥夺/复氧(oxygen glucose deprivation/reoxygenation,OGD/R)模型。方法 从SD乳鼠脑组织中分离海马组织,制备海马神经元悬液,通过抑制胶质细胞数量,将海马神经元悬液分为无抑制、部分抑制和完全抑制组,经镜下观察细胞形态,确定原代海马神经元的最佳培养条件。在此基础上,将海马神经元进行糖氧剥夺0.5、1和1.5 h,再分别复氧0、24、36和48 h,ELISA法检测培养液中乳酸脱氢酶(lactic dehydrogenase,LDH)含量,CCK-8法测定神经元存活率,以LDH含量较高,对神经元存活率影响较小的条件为原代海马神经元OGD/R模型的最佳建模条件。结果 部分抑制胶质细胞时,神经元纯度较高,生长状态均一良好,胶质细胞与海马神经元数量比约1∶1,可保留胶质细胞与神经元间的结构和功能联系。与OGD 1 h组比较,OGD 1 h/R 36 h时,LDH含量显著升高(t=4.31,P <0.05),神经元存活率差异无统计学意义(t=1.99,P> 0.05)。结论 获得了胶质细胞与神经元数量约为1∶1的原代海马神经元,且生长状态良好。OGD 1 h/R 36 h条件下建立的OGD/R模型,神经元树突缩短,活力受损,存活率较高。本研究为后续相关疾病的发病机制深入研究奠定了基础。Objective To establish an improved model of oxygen glucose deprivation/reoxygenation(OGD/R)in primary hippocampal neurons.Methods Hippocampal neurons were isolated from brain tissue of SD neonatal rat,and neuron suspension was prepared.By inhibiting the number of glial cells,the neuron suspension was divided into three groups,and no inhibition,partial inhibition and complete inhibition,the cell morphology was observed under microscope to determine the optimal culture conditions for primary hippocampal neurons.Based on this condition,the hippocampal neurons were deprived of glucose and oxygen for 0.5,1 and 1.5 h,and then reoxygenated for 0,24,36 and 48 h,separately.The content of lactic dehydrogenase(LDH)in culture medium was detected by ELISA,and the survival rate of neurons was measured by CCK-8 assay.The condition with higher LDH content and less influence on survival rate was the optimal condition for OGD/R model of primary hippocampal neurons.Results When the glial cells were partially inhibited,the purity of neurons was high with uniform growth status.The ratio of glial cells to hippocampal neurons was about 1:1,which preserved the structural and functional connection between glial cells and neurons.Compared with OGD 1 h group,the LDH content increased significantly at OGD 1 h/R 36 h(t=4.31,P<0.05),while there was no significant difference in neuronal survival rate(t=1.99,P>0.05).Conclusion Primary hippocampal neurons with about 1:1 number of glial cells and neurons were obtained with good growth status.The OGD/R model established under the condition of OGD 1 h/R 36 h has relatively high survival rate with shortened dendrites and impaired vitality,which lays a foundation for the further study of the pathogenesis of related diseases.

关 键 词：海马神经元 原代 糖氧剥夺/复氧模型 乳酸脱氢酶 细胞存活率 

分 类 号：R338[医药卫生—人体生理学]