二次分层离心纯化成年大鼠背根节神经元的方法  

A method to purify neurons in dorsal root ganglion of the adult rat via secondary layered centrifugation

作　　者：赵久红[1,2] 董栩[2] 张显芳[1,2] 张海英[1,2] 谭晓红[2] 张全鹏[1,2] 黄奕弟[1,2] 马志健[1,2] 易西南[1,2] 

机构地区：[1]海南医学院人体解剖学教研室,海口571199 [2]海南医学院神经科学研究所,海口571199

出　　处：《神经解剖学杂志》2016年第4期436-440,共5页Chinese Journal of Neuroanatomy

基　　金：国家自然科学基金资助(81130158;81460196);海南省自然科学基金(814299);海南省的教育厅科研项目(Hjkj2013-32)

摘　　要：目的:建立一种长时程的有效获得高度纯化成年大鼠背根节(dorsal root ganglions,DRG)神经元的方法。方法:将SD大鼠的DRG剥膜、消化制成单细胞悬液,牛血清白蛋白(bovine serum albumin,BSA)分层离心去除大部分非神经元细胞后接种于多聚赖氨酸处理的盖玻片上;培养1 d后,胰酶消化再次制成细胞悬液,BSA二次分层离心,再次接种于多聚赖氨酸处理的盖玻片上。BSA二次分层离心后的神经元为实验一(T1组)、单次离心的神经元为实验组二(T2组)、未经离心处理的神经元为对照组(C组)。各组除离心次数外,其余各方法相同。相差显微镜下观察上述各组培养神经元,结合βtubulinⅢ免疫荧光组化染色及MTT分析检测神经元纯化效果及细胞活力。结果:培养3 d后,T1组神经元比例达88.43±6.13%,较T2组、C组显著增高,差别具有统计学意义(P<0.05);MTT的结果显示与T2组、C组比较,T1组神经元的相对活力略微下降,但无统计学差异(P>0.05)。结论:二次纯化法是一种简单有效的成年大鼠DRG神经元纯化方法。Objective： To establish a long-term and effective purification method for the neurons in the dorsal root ganglion（ DRG） of adult rats. Method： The DRG harvested from the adult rats were stripped and digested into cell suspension under aseptic conditions. Before plated,neurons were purified for the first time via centrifugation with bovine serum albumin（ BSA） to deplete most non-neuron cells. After 1d in culture,neurons（ ganglion cells） were re-digested with trypsin,and neurons were purified for the second time via centrifugation with BSA. Neurons were divided into 3groups： group T1（ neurons with twice centrifugations）,group T2（ neurons with once centrifugation） and group C（ neurons without any centrifugation）. At different time points,neurons were observed under a phase contrast microscope. After 3 d in culture,immunofluorescence histochemical staining and MTT assay were performed. Results： Neurons purification rate in group T1 was 88. 43 ± 6. 13%,significantly higher than that in group T2 and group C（ P〈0. 05）. MTT assay showed the difference in neuron relative activity among group T2,group C and group T1 was not significant（ P〉0. 05）. Conclusion： Our results suggest that secondary layered centrifugation is a simple and effective method for purifying the DRG neurons of adult rats.

关 键 词：DRG 神经元培养 BSA 离心 纯化 大鼠 

分 类 号：R338[医药卫生—人体生理学]