新生大鼠海马神经元体外氧-糖剥夺再灌注模型的建立

Establishment of the model of oxygen-glucose deprivation and reperfusion in hippocampus neurons from neonatal rat in vitro

目的体外培养新生大鼠海马神经元,建立神经元氧-糖剥夺再灌注(OGD/R)模型,为临床进一步研究新生儿缺氧缺血性脑损伤奠定基础。方法以L-多聚赖氨酸和鼠尾胶原作为海马神经元体外培养的生长基质,分离24h内新生SD大鼠海马,胰蛋白酶水浴振荡消化获得单个细胞,采用Neurobasal维持培养基更继续培养。培养7d后将神经元细胞培养基更换为无糖缺血液,置于95%高纯度氮气(N2)三气培养箱内缺氧处理2h。将其取出更换为正常培养基继续培养24h,构建神经元OGD/R模型。然后,采用抗微管相关蛋白2-5-异硫氰酸荧光素(anti-MAP2-FITC)标记神经元,于荧光显微镜下观察建模前与建模后神经元的形态学改变,采用细胞活性检测试剂盒(CCK-8)检测微管相关蛋白(MAP)2阳性细胞存活率,对OGD/R模型进行评价。结果荧光显微镜下观察到正常情况下神经元细胞形态结构完整,树突及轴突舒展,交织成网状。建模后,神经元突触回缩,细胞崩解,网状结构被破坏。CCK-8检测结果显示,建模后神经元细胞存活率降低。结论此方法体外培养神经元细胞纯度在95%以上;使用无糖神经元细胞缺血液联合高纯度N2培养2h,再更换为正常培养基培养的方法,可成功建立神经元细胞的OGD/R模型。

Objective To culture primary hippocampus neurons and establish a better oxygenglucose deprivation and reperfusion（OGD/R）model for further research on neurons in vitro.Methods L-poly lysine and rat tail collagen were used as the growth mediuminvitro culture of neurons,then hippocampal tissues were isolated from the newborn SD rats（within 24 hours of the birth）,digested by single trypsin and bath shock,and made into cell suspension.The suspension was cultured with nutrient solution.7days later,to replace the medium with sugar free blood,and expose neurons to hypoxia in a three gas incubator containing 95% nitrogen（N2）for 2hours,then cells were returned to former incubator under normoxic conditions for 24 hours to build a OGD/R model.Evaluating the OGD/R model with observing the morphological changes of neurons and the cell viability with anti-microtubule-associated protein-fluorescein-5-isothiocyanate（anti-MAP2-FITC）immunofluorescent staining and cell activity assay kit cell counting kit-8（CCK-8）before and after the establishment of OGD/R model.Results With a fluorescence microscope,neuronal dendrites and axons stretched and overlapped into a network under normal circumstances were observed.After the establishment of OGD/R model,neuronal synapses retracted,cell disintegrated,reticular structure was destroyed and a decrease in neuronal viability was found by CCK8 test.Conclusions The neurons obtained by the aboved method were in good growth state,and the purity was higher than 95%.The OGD/R model of hippocampus neurons was successfully established by the way of replacing the normal medium with the sugar free medium combined with high purity N2 for 2hours from neonatal rat in vitro.