大鼠背根神经节神经元原代培养中纯化方法的改良  

Improvement of the purification methods for primary culture of neurons from dorsal root ganglion of rats

作　　者：李晓丽[1] 张石波[2] 孙晓婷 易剑锋 郭可盈 高小东 李人杰 强亮 丁斐[2] 施海燕 

机构地区：[1]南通大学医学院病理生理学系,南通226001 [2]江苏省神经再生重点实验室

出　　处：《南通大学学报（医学版）》2015年第4期257-260,254,共5页Journal of Nantong University(Medical sciences)

基　　金：国家自然科学基金资助项目(81301330;81371389;31371078);南通大学博士启动基金项目(13B30);南通大学大学生创新计划项目(2015084);江苏省基础医学优势学科建设工程资助项目(PAPD)

摘　　要：目的 :改进大鼠背根神经节神经元原代培养中的纯化方法以提高细胞纯度。方法 :常规取材并酶解消化大鼠背根神经节神经元,应用牛血清白蛋白溶液对细胞进行密度梯度离心初步分离细胞,再应用差速贴壁法去除非神经元细胞,最后应用阿糖胞苷进一步纯化细胞。纯化后应用显微镜观察细胞形态,采用β-tubulinⅢ免疫细胞化学染色方法进行纯度鉴定,应用CCK-8检测细胞活力。结果:未改良组获得纯化的背根神经节神经元的培养周期长达10 d,而改良组缩短为5 d。相差显微镜可见改良组神经胶质细胞明显减少。改良组的β-tubulinⅢ免疫细胞化学染色阳性细胞比例显著增加,计数结果显示改良组神经元纯度达到93%,未改良组纯度仅达到68%,差异有统计学意义(P<0.05)。CCK-8检测结果显示改良组的细胞活力高于未改良组,差异有统计学意义(P<0.05)。结论 :纯化方法改良后可以在更短的培养周期内获得纯度更高、细胞活力更好的大鼠原代背根节神经元。Objective: Improve the purification methods for primary culture of rat dorsal root ganglion(DRG) neurons to increase cell purity. Methods: DRG was routinely dissociated from rats and digested by enzymes. Cell prefractionation was performed by density-gradient centrifugation applying 15% bovine serum albumin(BSA) solution,and non-neuronal cells were removed by the differential velocity adherent technique, lastly Ara-C was used to purify neurons furtherly. The morphology of DRG neurons was observed under microscope. Immunocytochemical staining for β-tubulin Ⅲ was applied to identify neuron purity,and cell viability was examined by CCK-8 analysis. Results: The culture cycle to gain high purified DRG neurons was shortened from 10 days to 5 days after improvement of the purification methods. It was observed that the glial cells were obviously decreased in improved group under phase-contrast microscope. The ratio of β-tubulin Ⅲ immunocytochemical staining positive cells in improved group was evidently increased to 93%, and that was only 68% in unimproved group, the data difference was of statistical significance(P<0.05). CCK-8 assay showed that cell viability in improved group was higher than that in unimproved group, the data difference was of statistical significance( P < 0. 05). Conclusions : After improvement of purification methods, rat primary culture DRG neurons with higher purity and preferable cell viability are obtained in shorter culture cycle.

关 键 词：背根神经节神经元 细胞培养 纯化方法 大鼠 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]