刘检;王宇红;凌佳;刘林;孟盼;杨蕙;
LIU Jian;WANG Yuhong;LING Jia;LIU Lin;MENG Pan;YANG Hui;First Affiliated Hospital of Hunan University of Chinese Medicine;Hu'nan Key Laboratory of Power and Innovative Drugs State Key Laboratory of Ministry Training Bases;

• 1:湖南中医药大学第一附属医院
• 2:湖南省中药粉体与创新药物省部共建国家重点实验室培育基地

摘要(Abstract)：

目的建立一种优化的大鼠小胶质细胞(microglia,MG)原代培养及纯化方法,采用高内涵细胞成像分析技术(high content analysis,HCA)对其进行细胞鉴定。方法取1 d的新生SD大鼠,解剖显微镜下分离大脑皮质,机械剪碎成1 mm³组织块,加入质量分数为0. 125%的胰蛋白酶和质量分数为0. 1%的胶原酶(体积比为1∶1)消化10 min后进行体外培养,分别观察接种5、7 d后小胶质细胞基本形态结构;采用低速震摇+温和消化+差速贴壁的三联法纯化细胞,分别观察纯化1、3、6 d后小胶质细胞基本形态结构;采用HCA技术对小胶质细胞进行其特异性抗体(ionized calcium binding adapter molecule 1,Iba-1)免疫细胞化学染色鉴定。结果经优化法培养的小胶质细胞,数量多、纯度高、活性好,细胞胞体狭长,突起形状不规则,呈单极、双极、蜘蛛状或其它分枝状,且分别可见静息态与活化态的细胞,并呈现典型的小胶质细胞特征和良好的生长状态;经优化法培养的小胶质细胞,采用HCA进行Iba-1免疫细胞化学染色鉴定,MG纯度质量分数达98%以上。结论建立了一种小胶质细胞原代培养及纯化方法,同时采用HCA技术成功对其进行细胞鉴定,为后续小胶质细胞相关体外研究奠定了良好的实验基础。
Objective To establish an optimized method of primary cultivation and purification for Sprague Dawley( SD) rat microglias and to identify the cell by high content analysis. Methods Microglias were isolated from neonatal SD rat for one day under dissecting microscope,digested by 0. 125% trypsin-0. 1%collagenase( volume ratio 1∶ 1) after mechanical isolation with 1 mm3 tissue fragments,and then seeded into cell culture flask to observe its basic morphology structure after planted 5 days and 7 days. The triple methods of lowvelocity shaking,mild digestion combined with differential adhesion were employed to purify microglias,and basic morphology structure of microglias were observed after purified 1 day,3 day and 6 day,respectively. Immunocytochemistry staining for Iba-1 was performed to identify microglias by high content analysis technology. Results Microglias for SD rat were successfully cultured by optimized method with high quantity,purity and activity. The cultured microglias with elongated cell body and monopole,duotriode,spider or other branch-shaped cell processes were in good growth condition and typical microglias morphological feature of resting-statea or activated state. The immunocytochemistry staining for Iba-1 showed that the purity of microglias was over 98% by optimized culture method and high content analysis.Conclusion An optimized method of primary cultivation and purification for SD rat microglias is successfully established with advantages of high efficiency,easy operation,high cell quantity,purity and activity. High content analysis technology is successfully employed to identify microglias,which provides a good experimental base for subsequent research of microglias in vitro.

关键词(KeyWords)： 小胶质细胞;原代培养;纯化;鉴定;小胶质细胞特异性抗体(Iba-1);高内涵细胞成像分析技术(HCA)
microglias;primary culture;purification;identification;ionized calcium binding adapter molecule 1(Iba-1);HCA

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金资助项目(81403379,81573965,81603604);; 湖南省自然科学基金资助项目(2017JJ3241);; 湖南省教育厅科学研究一般项目(17C1229)

作者(Authors): 刘检;王宇红;凌佳;刘林;孟盼;杨蕙;
LIU Jian;WANG Yuhong;LING Jia;LIU Lin;MENG Pan;YANG Hui;First Affiliated Hospital of Hunan University of Chinese Medicine;Hu'nan Key Laboratory of Power and Innovative Drugs State Key Laboratory of Ministry Training Bases;

DOI: 10.14066/j.cnki.cn21-1349/r.2018.11.010

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