阿糖胞苷对原代培养的大鼠海马和大脑皮层神经元生物学特性的影响  

Effects of cytosine arabinoside on biological properties of primary cultured hippocampal and cortical neurons in rats

作　　者：戴嘉华 孙卫文[1] 赖梅菁 邱美茜 张文浩[1,2] 徐恩 詹丽璇[1] DAI Jiahua;SUN Weiwen;LAI Meijing;QIU Meiqian;ZHANG Wenhao;XU En;ZHAN Lixuan(Institute of Neurosciences,Department of Neurology,The Second Affiliated Hospital of Guangzhou Medical University,Guangzhou 510260,Guangdong,China;Key Laboratory for Regenerative Medicine of Ministry of Education,Jinan University,Guangzhou 510632,Guangdong,China)

机构地区：[1]广州医科大学附属第二医院神经内科广州医科大学神经科学研究所,广东广州510260 [2]暨南大学再生医学教育部重点实验室,广东广州510632

出　　处：《广州医科大学学报》2023年第6期1-10,共10页Academic Journal of Guangzhou Medical University

基　　金：国家自然科学基金面上项目(82271330);广东省自然科学基金(2023A1515010210);再生医学教育部重点实验室(暨南大学)开放基金(ZSYXM202210)。

摘　　要：目的:探讨阿糖胞苷(Ara-C)不同浓度、不同介入时间以及不同处理时长对原代培养的大鼠海马和大脑皮层神经元纯度及活性的影响。方法:取孕16~18 d的胎鼠海马及皮层组织进行原代神经元培养,培养后48 h分别加入浓度为0、2.5、5、7.5、10μmol/L的Ara-C处理24 h,检测不同浓度的Ara-C对原代神经元生物学特性的影响;培养后24、48、72、96及120 h,加入5μmol/L的Ara-C处理24 h,检测Ara-C不同介入时间对原代神经元生物学特性的影响;培养后48 h,加入5μmol/L的Ara-C处理0、6、12、24、48 h,检测Ara-C不同处理时长对原代神经元生物学特性的影响。培养后第8天倒置显微镜下观察细胞的形态及生长状况,免疫荧光检测微管相关蛋白2(MAP2)、神经纤维酸性蛋白(GFAP)的表达,并计算MAP2阳性细胞占细胞总数的比例。使用细胞计数试剂盒8(CCK-8)检测神经元细胞活性。结果:与对照组(0μmol/L)相比,Ara-C浓度为5μmol/L组海马神经元纯度达89.8%、细胞活性为48.6%。与对照组相比,种板后48 h加入Ara-C获得的海马神经元纯度为96.2%,细胞活性为44.8%。与对照组(0 h)相比,Ara-C处理12 h组获得的海马神经元的纯度为90.8%,细胞活性为47.5%。在大鼠原代培养的皮层神经元中,与对照组(0μmol/L)相比,给予不同浓度的Ara-C(2.5、5、7.5、10μmol/L)处理,原代皮层神经元纯度的差异无统计学意义(P>0.05)。与对照组相比,给予不同介入时间(24、48、72、96、120 h)的Ara-C处理,皮层神经元纯度的差异无统计学意义(P>0.05)。与对照组(0 h)相比,给予不同时长(6、12、24、48h)的Ara-C处理,仅Ara-C处理24 h后皮层神经元纯度稍增高(P<0.05)。结论:在大鼠原代海马神经元的早期培养过程中,种板后48 h加入5μmol/L Ara-C处理12 h得到的海马神经元的纯度与活性最佳,而在大鼠原代皮层神经元培养中无需添加Ara-C即可获得较高纯度和活性的神经元。Objective:To determine the optimal dosage,intervention interval,and administration duration of cytosine arabinoside(Ara-C)on the purity and activity in primary cultured hippocampal and cortical neurons in rats.Methods:Hippocampal and cortical tissues of fetal rats at 16-18 days of gestation were taken for primary neuron culture.Firstly,to examine the effects of different concentrations of Ara-C on the biological properties of neurons,after 48 h of culture,Ara-C with concentrations of 0,2.5,5,7.5 and 10μmol/L,respectively,was added and treated for 24 h.Secondly,to detect the effects of different intervention times of Ara-C on the biological properties of neurons,5μmol/L Ara-C was added at 24,48,72,96 and 120 h,respectively,and treated for 24 h.Thirdly,to investigate the effects of different administration durations of Ara-C on the biological properties of neurons,after 48 h of culture,5μmol/L Ara-C was treated for 0,6,12,24 and 48 h,respectively.The morphology and growth of neurons were observed using the inverted microscope at 8 days of culture.Immunofluorescence was used to detect the expression of microtubule-associated protein 2(MAP2)and glial fibrillary acidic protein(GFAP).And the proportion of MAP2-positive cells to the total number of cells was calculated.Lastly,the viability of primary culture neurons was identified by Cell Counting Kit-8 assays(CCK-8).Results:Compared with the control group(0μmol/L),the purity and viability of primary culture hippocampal neurons with 5μmol/L Ara-C were 89.8%and 48.6%,respectively.Compared with the control group,the purity of hippocampal neurons cultured with Ara-C at 48 h after culture was 96.2%,and the cell viability was 44.8%.Compared with the control group(0 h),the purity of hippocampal neurons administrated with Ara-C for 12 h was 90.8%,and the viability of neurons was 47.5%.In rat primary cultured cortical neurons,compared with the control group(0μmol/L),the concentrations of Ara-C(0,2.5,5,7.5 and 10μmol/L)had no effect on the purity of cortical primary neuro

关 键 词：海马和皮层神经元 原代培养 阿糖胞苷 

分 类 号：R338.2[医药卫生—人体生理学]