新生SD大鼠大脑皮质神经元的原代培养及鉴定

Primary culture and identification of neurons from cerebral cortex of newborn Sprague-Dawley rats

背景:培养神经元的质量是研究中枢神经系统疾病的关键所在,但培养方法一直没有统一的标准。目的:探究取材部位及阿糖胞苷处理对原代培养神经元活性、纯度及成熟度的影响。方法:选取新生24 h内的SD乳鼠,取大脑皮质表面2.0-3.0 mm处细胞,培养后24,48,72 h,用5μmol/L或10μmol/L阿糖胞苷处理48 h;另取全部大脑皮质细胞,培养后48 h用5μmol/L阿糖胞苷处理48 h。培养至第7天,倒置显微镜下观察细胞形态,采用CCK-8法检测阿糖胞苷对神经元活性的影响,免疫荧光和Western blot检测神经元的纯度及成熟度。结果与结论:①倒置显微镜:10μmol/L阿糖胞苷24,48,72 h组神经元密度分别低于同时间下的5μmol/L阿糖胞苷组,该6组神经元突起相互之间均可连接成紧密的网状结构,神经元胞体丰满并充满折光特性;全皮质组细胞密度低于正常对照组,并可明显观察到非神经元;②CCK-8检测:各阿糖胞苷组细胞活性均低于正常对照组(P<0.001),10μmol/L阿糖胞苷24,48,72 h组神经元活性低于同时间下的5μmol/L阿糖胞苷组(P<0.05),5μmol/L阿糖胞苷24 h组神经元活性低于5μmol/L阿糖胞苷48,72 h组(P<0.01);③免疫荧光染色:各阿糖胞苷组神经元纯度大于正常对照组、全部皮质组(P<0.001),各阿糖胞苷组神经元纯度无差异;④Western blot检测神经元特异性烯醇化酶蛋白表达:各阿糖胞苷组神经元成熟度高于正常对照组(P<0.05),其中以5μmol/L阿糖胞苷48 h组成熟度最高;⑤结果表明:取新生24 h内大鼠大脑皮质表面2.0-3.0 mm处皮质,培养后48 h加入5μmol/L阿糖胞苷处理得到的神经元活性、成熟度和纯度较好。

BACKGROUND:The quality of cultured neurons is the key to the study of central nervous system diseases,but there has been no unified standard for the methods used.OBJECTIVE:To explore the effects of best selection site and cytarabine treatment conditions on the viability,purity and maturity of primary cultured neurons.METHODS:The cells were obtained at 2.0-3.0 mm on the surface of the cerebral cortex of Sprague-Dawley rats within 24 hours of the newborn,and treated with cytarabine at a concentration of 5μmol/L or 10μmol/L at 24,48,and 72 hours after culture.In addition,the whole cerebral cortex was removed for primary culture and treated with cytarabine at a concentration of 5μmol/L for 48 hours at 48 hours after culture.At 7 days after culture,the morphology of neuron was observed using inverted microscope and the effect of cytarabine on neuronal viability was detected by CCK-8 assay.The purity and maturity of neurons were assessed by immunofluorescence and western blot assay.RESULTS AND CONCLUSION:(1)Inverted microscope:The density of neurons in the 10μmol/L cytarabine group at 24,48,and 72 hours was lower than that in the 5μmol/L cytarabine group at the same time point,and the neuronal processes in the six groups could be connected to each other to form a dense network structure.The neuron cell body was plump and full of refractive properties.The cell density of the whole cortex group was lower than that of the normal control group,and non-neuronal cells could be clearly observed.(2)CCK-8 assay:The cell viability of each cytarabine group was lower than that of the normal control group(P<0.001),and the neuronal viability of the 10μmol/L cytarabine 24-,48-,and 72-hour groups was lower than that of 5μmol/L cytarabine group at the same time point(P<0.05).The neuronal viability in the 5μmol/L cytarabine 24-hour group was lower than that in the 5μmol/L cytarabine 48-and 72-hour group(P<0.01).(3)Immunofluorescence staining:The purity of neurons in each cytarabine group was greater than that in the normal control group and all cortical groups(P<0.001),and there was no difference in the purity of neurons in each cytarabine group.(4)Western blot assay detection of neuron-specific enolase protein expression:The neuron maturity of each cytarabine group was higher than that of the normal control group(P<0.05),and the 5μmol/L cytarabine 48-hour group had the highest maturity.(5)The results showed that the cortex at 2.0-3.0 mm on the surface of the rat cerebral cortex within 24 hours of the newborn treated with 5μmol/L cytarabine at 48 hours after culture can obtain better neuronal viability,maturity and purity.