• 首页关于本刊期刊订阅编委会作者指南过刊浏览
黎晓慧,李明学,黄鑫,张志成,袁园,代解杰.2019.树鼩原代小胶质细胞的分离培养、纯化与鉴定.动物学杂志,54(3):445-450.
树鼩原代小胶质细胞的分离培养、纯化与鉴定
Isolation and Identification of Primary Microglia from Tree Shrew
投稿时间:2018-12-28  修订日期:2019-04-22
DOI:10.13859/j.cjz.201903015
中文关键词:  树鼩  原代小胶质细胞  直立手拍法  温和胰酶消化法  恒温振荡法
英文关键词:Tree shrew  Primary microglia  Upright hand clapping method  Mild digestion with trypsin method  Constant temperature oscillation method
基金项目:云南省科技人才和平台计划项目(No. 2017HC019),云南省重大科技专项(No. 2017ZF007),国家科技支撑计划项目 (No. 2014BAI01B00),云南省联合支持国家计划项目(No. 2015GA009)
作者单位E-mail
黎晓慧 ①② 中国医学科学院/北京协和医学院 医学生物学研究所 云南省重大传染病疫苗研发重点实验室 昆明 650118 ② 昆明医科大学 昆明 650500 1760440328@qq.com 
李明学 ①② 中国医学科学院/北京协和医学院 医学生物学研究所 云南省重大传染病疫苗研发重点实验室 昆明 650118 ② 昆明医科大学 昆明 650500 1363251417@qq.com 
黄鑫 ①② 中国医学科学院/北京协和医学院 医学生物学研究所 云南省重大传染病疫苗研发重点实验室 昆明 650118 ② 昆明医科大学 昆明 650500 642975085@qq.com 
张志成 ①② 中国医学科学院/北京协和医学院 医学生物学研究所 云南省重大传染病疫苗研发重点实验室 昆明 650118 ② 昆明医科大学 昆明 650500 373773431@qq.com 
袁园 ①② 中国医学科学院/北京协和医学院 医学生物学研究所 云南省重大传染病疫苗研发重点实验室 昆明 650118 ② 昆明医科大学 昆明 650500 1211092538@qq.com 
代解杰 ①② 中国医学科学院/北京协和医学院 医学生物学研究所 云南省重大传染病疫苗研发重点实验室 昆明 650118 ② 昆明医科大学 昆明 650500 djj@imbcams.com.cn 
摘要点击次数: 206
全文下载次数: 181
中文摘要:
      本文旨在建立树鼩(Tupaia belangeri)小胶质细胞原代培养及纯化的方法,为利用新型实验动物树鼩进行相关研究工作提供实验材料。将新生树鼩大脑皮质机械分离,皮质组织块用胰蛋白酶消化后制成细胞悬液;培养9 ~ 10 d后,分别采用直立手拍法、温和胰酶消化法以及恒温振荡法分离纯化树鼩小胶质细胞,通过差速贴壁进一步纯化。荧光显微镜下,利用小胶质细胞的特异性标记物CD11b抗体进行鉴定。结果显示,小胶质细胞分离培养第3天时呈静息状态,表现为梭形、杆状、分支状等不规则形态。细胞免疫荧光CD11b呈阳性。不同纯化方法细胞免疫荧光并计数显示,直立手拍法所获得的细胞产量明显高于恒温振荡法(P < 0.05),细胞阳性率(> 96%)明显高于温和胰酶消化法(> 90%,P < 0.05)。直立手拍法可获得产量及纯度高的树鼩原代小胶质细胞。
英文摘要:
      The aim of this study is to establish a method for purifying and culturing cerebral microglial cells of tree shrew (Tupaia belangeri), providing new experimental materials for future research. Newborn tree shrews were used in this experiment. The cortex of cerebrum was isolated and disaggregated by trypsin digestion. The microglia cells were purified by three methods after culturing for 9﹣10 days: upright hand clapping, mild digestion with trypsin and constant temperature oscillation. Meanwhile,we also used differential attachment method to further purify cells. At last, the number of purified cells was counted using a cell counter. Purified microglial cells were labeled with specific marker CD11b and observed with fluorescence microscope. The results showed that microglial cells were in a resting state on the third day of isolation and culture, with irregular shapes such as spindle, rod and branch (Fig. 2). Purified microglial cells were positively labeled with specific marker CD11b as revealed by fluorescence microscopy (Fig. 3). Data statistics was performed using SPSS statistical software. Analysis of cellular immunofluorescence intensities and counting of cells purified by different purification methods showed that the cell yield obtained by the upright hand clapping method was significantly higher than that obtained by constant temperature oscillation method, and the positive cell rate was significantly higher than that obtained by mild trypsin digestion method (Fig. 4). Primary microglial cells of tree shrews with high yield and purity were obtained by upright hand clapping.
附件
查看全文  查看/发表评论  下载PDF阅读器