一种改良后的大脑皮质神经元细胞的原代培养模型建立及鉴定  

Establishment and identification of an improved primary culture model of cerebral cortical neurons

作　　者：张磊[1,3] 韩延峰 戴朝 李阳[1] 胡玉奇 陈富雷 赵冬 ZHANG Lei;HAN Yanfeng;DAI Chao;LI Yang;HU Yuqi;CHEN Fulei;ZHAO Dong(Department of Neurosurgery,First Affiliated Hospital,School of Medicine,Shihezi University,Shihezi,Xinjiang 832000,China;Department of Xinjiang Production and Construction Corps Tenth Division Disease Prevention and Control Center,Beitun,Xinjiang 836000,China;Department of Xinjiang Production and Construction Corps Tenth Division Beitun Hospital,Beitun,Xinjiang 836000,China)

机构地区：[1]石河子大学医学院第一附属医院神经外科,新疆石河子832000 [2]新疆生产建设兵团第十师疾病预防控制中心,新疆北屯836000 [3]新疆生产建设兵团第十师北屯医院,新疆北屯836000

出　　处：《石河子大学学报（自然科学版）》2022年第1期100-105,共6页Journal of Shihezi University(Natural Science)

基　　金：国家自然科学基金项目(81960222);新疆兵团中青年科技创新领军人才(2020CB011);石河子大学国际科技合作促进计划项目(GJHZ201704)。

摘　　要：目的在传统的大脑皮质神经元的提取和培养方法基础上进行优化改良以获得高收率、高纯度、高质量、低死亡率、背景干净的皮质神经元细胞。方法分别采用传统的含EDTA的0.25%Trypsin分离培养的皮质神经元,与改良后用混有Tryple Express和DnaseI的消化液,经弹拨分散、手法震荡,提前一次离心后种植培养的皮质神经元。接种后的第1、3、5、6、7 d,显微镜观察细胞形态,第7 d用免疫荧光法对神经元细胞进行鉴定以及检测细胞培养液中乳酸脱氢酶(LDH)的释放量。结果与传统的胰酶提取方法相比,改良后获得的皮质神经元生长状态更优,突触发育良好,双极神经元形态典型,杂质细胞及碎片含量少,背景干净,且具有高纯度和高存活率的特性。结论本研究在组织细胞分离消化,弹拨分散、手法震荡,提前一次离心,多重细节方面进行了探索与改良后成功建立了一种结果稳定,简单可行的皮质神经元原代培养方法,是研究神经科学领域较为理想的模型。Objective Based on the traditional methods of cortical neuron extraction and culture, we optimized and improved the methods to obtain cortical neuron cells with high yield, high purity, high quality, low mortality and clean background.Methods Cortical neurons were isolated and cultured with traditional 0.25%Trypsin containing EDTA,and then transplanted with modified digestive juice mixed with Tryple Express and DnaseI.The cultured cortical neurons were dispersed by plastering, disturbed by technique, and centrifuged once in advance.On the 1 st, 3 rd, 5 th, 6 th and 7 th day after inoculation, the morphology of the cells was observed by inverted microscope.On the 7 th day of culture, the neurons were identified by immunofluorescence and the release of lactate dehydrogenase(LDH) from the cell culture medium was detected.Results Compared with the traditional trypsin extraction method, the improved cortical neurons have better growth state, good synaptic development, typical bipolar neuron morphology, less magazine cells and fragments, clean background and high purity and high survival rate. Conclusion In this study, a simple and feasible primary culture method of cortical neurons with stable results was successfully established after exploration and improvement in the aspects of tissue and cell separation and digestion, plicking and dispersing, manipulative oscillation, one centrifugation in advance, and multiple details, which is an ideal model for research in the field of neuroscience.

关 键 词：大脑皮质神经元 Tryple Express 神经元特异性烯醇化酶 乳酸脱氢酶 免疫荧光染色 

分 类 号：R331[医药卫生—人体生理学]