一种稳定高效的新生大鼠皮质神经元原代培养方法  

A method for primary culture of newborn rat cortical neurons with high viability

作　　者：马万[1,2] 袁文俊[1] 焦英甫[3] 王春生[1] 陈桂英[1] 孟尽海[2] 

机构地区：[1]宁夏医科大学,银川750004 [2]宁夏医科大学总医院麻醉科,银川750004 [3]第二军医大学第三附属医院疼痛科,上海200438

出　　处：《神经解剖学杂志》2015年第4期510-514,共5页Chinese Journal of Neuroanatomy

基　　金：宁夏自然科学基金(NZ11183)

摘　　要：目的:建立一种稳定、高效、简单可行的新生大鼠大脑皮质神经元原代培养方法。方法:取新生24 h内的SD大鼠,分离皮质,采用木瓜蛋白酶消化、实验前多聚赖氨酸铺板、不同机械吹打次数及细胞接种2 h后全量换为添加B-27的Neurobasal A培养基的培养方法,MTT比色法分析检测神经元细胞成活率;于不同时间在倒置相差显微镜下观察细胞的生长状态;利用神经元特异性标志物微管蛋白相关标志物2(microtubule associated protein 2,MAP2)鉴定神经元纯度。结果:木瓜蛋白酶明显增加原代培养神经元细胞活力(P<0.01),采用适度的机械吹打、实验前多聚赖氨酸铺板及细胞接种2 h后全量换为添加B-27的Neurobasal A培养基,对神经细胞的活力没有明显的影响(P>0.05)。大部分皮质神经元具有典型的神经元形态特征,经MAP2免疫荧光技术鉴定神经元所占比例达95%以上。结论:该方法操作简单高效,方便可行,结果稳定。Objective： To establish a stable, effective, convenient and practicable method for primary culture of neu- rons from cerebral cortex of neonate rats. Methods： SD rats were taken within 24 hours after birth. Their cerebral cortex was separated and digested with papain, and then the neurons were planked with polylysine before experiment. After me- chanical treatment for different times and a 2 - hour of cell inoculation, the culture medium for the neurons was totally re- placed by Neurobasal A medium that contained B -27. The survival rates of neurons were detected by M＇I^F assay. The morphological changes of neuron cells were observed under light microscope at different time. Microtubule - associated protein - 2 （ MAlY2）, the specific maker of the neurons was used to identify the purity of neurons. Results： Papain can obviously increase the cell viability of the primary cultured neurons （ P 〈 0.01 ）. The method including mild mechanical treatment, plating with polylysine before experiment and replacement of the culture medium with Neurobasal A medium that contained B - 27 after a 2 - hour of cell inoculation, had no significant effect on the viability of neurons （ P 〉 0.05 ）. Most cortical neurons had typical neuron morphology. Fluorescence staining with MAP＇2 showed that neurons accounted for more than 95%. Conclusion： This method is simple, effective, convenient and reliable.

关 键 词：大脑皮质 神经元 原代培养 新生大鼠 

分 类 号：R741[医药卫生—神经病学与精神病学]