新生大鼠原代小胶质细胞分离培养方法的改良  

Modified method for cultivating primary microglia of neonatal rat

作　　者：钱凯 唐琳俊 王希[1] 杨坤[3] 张开鑫[1] 钱腾达[1] 马涛 石晶[1] 李立新[1] QIAN Kai;TANG Lin-jun;WANG Xi(Department of Neurosurgery,First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China)

机构地区：[1]南京医科大学第一附属医院神经外科,南京210029 [2]安徽省芜湖市第二人民医院神经外科 [3]南京脑科医院神经外科

出　　处：《临床神经外科杂志》2019年第1期1-5,共5页Journal of Clinical Neurosurgery

基　　金：国家自然科学基金面上项目(81171147);江苏省科教强卫工程医学重点人才研究基金(ZDRCA2016010);江苏省兴卫工程医学重点人才基金(RC201156)

摘　　要：目的研究改良体外小胶质细胞分离培养的方法及效果。传统法存在动物用量大、成本高且细胞培养时间长等局限。方法结合消化试剂组合和手摇震荡法培养分离小胶质细胞;同时采用Iba-1免疫荧光法进行细胞鉴定和纯度分析,台盼蓝染料法进行活力检测以及脂多糖刺激比较不同状态的小胶质细胞。结果改良法可在4 d内稳定获得小胶质细胞> 1. 0×10~6个/60 mm培养皿,纯度≥98%,活力> 98%,较传统法在动物用量、总成本和培养时间分别减少了2、3. 2、1. 5倍。结论改良法可减少动物用量、降低成本,缩短细胞培养时间;为研究小胶质细胞的生物学功能和中枢神经系统疾病机制建立了一种稳定、简便、高效的细胞模型。Objective Isolation and culture of microglia in vitro has been widely used to explore new therapeutic strategies for various central nervous system diseases,but the existing protocols need a large amount of animal,higher cost and long culture time.Methods Combination of a unique combination of digestive reagents and shaking by hand were used to isolate and purify microglia.The purity of isolated cells was identified by expression of Iba-1 utilizing immunofluorescence technique and the viability of microglia was determined with trypan blue exclusion.Meantime,comparing the different states of microglia using the lipopolysaccharide test.Results The present modified methodology steadily yielded 1.0×10^6 microglia per 60 mm dish with high purity(≥98%)and high survival rate( >98%)within four days.Compared to the existing protocols,the animal dosage,total cost and culture time were reduced by 2,3.2 and 1.5 times,respectively.Conclusion The improved method establishes a stable,simple and efficient cell culture model for studying the biological functions of microglia and elucidating the mechanisms of related CNS diseases.

关 键 词：小胶质细胞 原代培养 新生大鼠 

分 类 号：R65[医药卫生—外科学]