[1]仇海文,李 卉,刘振州,等.Ⅱ型胶原诱导的关节炎大鼠滑膜成纤维细胞的培养、纯化和鉴定[J].南京师范大学学报(自然科学版),2017,40(03):128.[doi:10.3969/j.issn.1001-4616.2017.03.019]
　Qiu Haiwen,Li Hui,Liu Zhenzhou,et al.Isolation,Culture and Identification of Fibroblast-Like Synoviocytesfrom Collagen-Induced Arthritis(CIA)Rats[J].Journal of Nanjing Normal University(Natural Science Edition),2017,40(03):128.[doi:10.3969/j.issn.1001-4616.2017.03.019]
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Ⅱ型胶原诱导的关节炎大鼠滑膜成纤维细胞的培养、纯化和鉴定()
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《南京师范大学学报》（自然科学版）[ISSN:1001-4616/CN:32-1239/N]

卷:
第40卷
期数:
2017年03期
页码:
128
栏目:
·生命科学·
出版日期:
2017-09-30

文章信息/Info

Title:
Isolation,Culture and Identification of Fibroblast-Like Synoviocytesfrom Collagen-Induced Arthritis(CIA)Rats
文章编号:
1001-4616(2017)03-0128-05
作者:
仇海文; 李 卉; 刘振州; 孙胜楠; 马雪梅; 刘 梅
南京师范大学生命科学学院,江苏省分子医学生物技术重点实验室,江苏 南京 210023
Author(s):
Qiu Haiwen; Li Hui; Liu Zhenzhou; Sun Shengnan; Ma Xuemei; Liu Mei
School of Life Sciences,Nanjing Normal University,Jiangsu Key Laboratory for Molecular and Medical Biotechnology,Nanjing 210023,China
关键词:
滑膜成纤维细胞; 胶原诱导的关节炎大鼠模型; 原代培养; 类风湿关节炎
Keywords:
synovial fibroblasts; collagen-induced arthritis rat model; primary culture; rheumatoid arthritis
分类号:
Q2-33
DOI:
10.3969/j.issn.1001-4616.2017.03.019
文献标志码:
A
摘要:
类风湿关节炎(Rheumatoid arthritis,RA)病人滑膜成纤维细胞(Fibroblast-like synoviocytes,FLS)是研究RA发病机制和进行RA药物筛选的最理想的细胞来源. 但是对于众多非医疗机构的高校和科研院所而言,由于RA病人滑膜组织不易获取而难以进行细胞水平的操作. 因此,建立胶原诱导的关节炎大鼠(Collagen-induced arthritis,CIA)模型,成功分离膝关节滑膜组织并进行滑膜成纤维细胞的原代培养和纯化,将有效解决RA FLS的来源问题,为后续实验奠定基础. 本实验通过皮内和皮下两次注射鸡Ⅱ型胶原诱导Wistar大鼠罹患关节炎,通过关节炎指数评分和膝关节HE染色明确CIA模型的建立; 分离膝关节滑膜组织,采用Ⅱ型胶原酶和无EDTA胰酶消化法分离细胞并进行传代培养,传至第三代时,细胞免疫荧光检测细胞中VCAM-1蛋白含量以鉴定细胞纯度. 结果显示,滑膜组织经过消化和培养后得到大量大鼠滑膜成纤维细胞; 在显微镜下观察所得细胞呈梭形树突状,具有成纤维样细胞特征; VCAM-1蛋白阳性细胞达98%以上,确证所分离细胞为滑膜成纤维样细胞,且纯度极高. 本研究建立了较简便快捷地获得大量滑膜成纤维细胞的方法,为RA的发病机制研究和药物防治提供了丰富的实验材料.
Abstract:
Fibroblast-like synoviocytes(FLS),which derived from rheumatoid arthritis(RA)patients,are the best cell source for the investigation of pathogenesis and treatment of RA. But it is difficult for some universities and institutions,to obtain human synovial tissue. So,it appears to be of essence to establish collagen-induced arthritis(CIA)rat model and there after obtain synovium for primary culture. The approach will be helpful to provide RA FLS efficiently,and lay the foundation for the follow-up experiment. Female Wistar rats were immunized intradermally with 1.5 mg native chicken collagen type II with an equal volume of Freund’s complete adjuvant. Seven days later,the rats were then given a subcutaneous booster injection of half the amount of CII emulsified in Freund’s incomplete adjuvant. When CIA model was established,the knee synovial tissue was separated and digested with type II collagenase and trypsin without EDTA. When the cells were cultured to the third generation,VCAM-1 protein in the cells was determined by immunostaining to identify cell purity. We obtained a large number of spindle-shaped cells. More than 98% of these cells were VCAM-1 positive,suggesting that these cells were rat synovial fibroblast-like cells in high purity. The establishment of a simple and rapid method for obtaining a large number of synovial fibroblasts will provides sufficient experimental materials for the pathogenesis and treatment of RA.

参考文献/References:

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备注/Memo

备注/Memo:
收稿日期:2016-11-15.
基金项目:国家自然科学基金面上项目(31171135)、江苏高校优势学科建设工程资助项目.
通讯联系人:刘梅,副教授,研究方向:类风湿关节炎发病机制和药物防治. E-mail:meiliunj@163.com
更新日期/Last Update: 2017-09-30