三种培养原代滑膜成纤维细胞方法的比较  

Comparison of Three Methods for Culturing Rheumatoid Arthritis Synovial Fibroblasts

作　　者：马莎[1] 林俊[1] 于亮[2] 晋松[1] 李芹[1] 张虹[1] 范宏涛[1] MA Sha;LIN Jun;YU Liang;JIN Song;LI Qin;ZHANG Hong;FAN Hong-tao(Dept. of Rheumatoid 驭Immunology,The First People’s Hospital of Yunnan Province,Kunming Yunnnan 650032;Dept. of Medical Genetics,Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College,Kunming Yunnan 650118,China)

机构地区：[1]云南省第一人民医院风湿免疫科,云南昆明650032 [2]中国医学科学院&北京协和医学院医学生物学研究所遗传室,云南昆明650118

出　　处：《昆明医科大学学报》2016年第6期52-56,共5页Journal of Kunming Medical University

基　　金：云南省科技厅-昆明医科大学联合专项基金资助项目(2011FB215)

摘　　要：目的比较3种体外分离培养原代类风湿关节炎滑膜成纤维样细胞（RASFs）的方法，寻求获得快速有效的培养途径．方法将来自关节镜RA滑膜切除术的滑膜组织分别采用胶原酶消化法、改良组织块法、双酶消化法进行培养．采用倒置相差显微镜观察细胞形态及生长特性，以Vimentin组织化学法进行鉴定．台盼兰计数培养14d后所得活细胞数目．结果3种原代分离培养方法均可成功培养出RASFs，符合RASFs的形态特征，Vimentin阳性细胞＞95%，其中：胶原酶消化培养法分离的RASFs16-20d细胞汇合度可达70%，约４周汇合度95%；改良组织块法分离的RASFs10～14d细胞汇合度可达70%以上；双酶消化法分离的RASFs约４周细胞汇合度达85%．处理相同数量滑膜组织获得活细胞数目比较，改良组织块法组为（1.60±0.08）×106个，胶原酶消化法组（1.41±0.08）×106个，双酶消化法组（1.19±0.05）×106个，差异有统计学意义（＜0.05）．培育原代RASFs细胞所需时间比较，胶原酶消化法需（267.50±16.58）min，双酶消化法需（183.75±11.08）min，改良组织块法需（149.10±13.71）min，差异有统计学意义（＜0.05）．结论改良组织块培养法能最大限度地利用滑膜组织，获得最多数量原代RASFs细胞，是建立体外RA细胞模型最高效、快捷的方法．Objective To compare the three kinds of methods for in vitro primary culturing of rheumatoid arthritis synovial fibroblast-like cells(RASFs), in order to get fast and effective culture methods. Methods Synovial tissue from RA synovial arthroscopic resection were treated by collagenase digestion method, modified tissue culture method, double enzyme digestion method respectively. By using an inverted phase contrast microscope, cell morphology and growth characteristics were observed and identified with vimentin staining. Trypan blue was used to count the number of living cells after culturing for 14d. Results The three primary methods could successfully isolate and culture RASFs,and RASFs met the morphological characteristics of vimentin-positive cells>95%,namely,the proportion of RASFs cell confluence was 70% after 16-20days by the collagenase digestion method,whose cell confluence proportion reached 95% after 4 weeks;and the cell confluence proportion was above 70% after 10-14days by modified tissue culture method,and the cell confluence proportion reached 85% after 4 weeks by the double enzyme digestion method. The comparison of the viable cells number cultured same number of synovial tissue by the three methods show the viable cells number cultured by the modified tissue culture method were(1.60±0.08) ×106, those by the collagenase digestion method were (1.41±0.08) ×106, those by the double enzyme digestion method were(1.19±0.05) ×106, which were with significant difference among them( <0.05).The comparison of incubation time of RASFs primary cells showed it took (267.50±16.58) mins by the collagenase digestion method, (183.75±11.08) mins by the double enzyme digestion method, and 149.10±13.71mins by the modified tissue culture method,with significant differences( <0.05) .Conclusion Modified tissue culture for RASFs is an efficient and fast culture method, the number and purity of RASFs can meet the requirements for biology experiments.

关 键 词：类风湿关节炎 滑膜成纤维细胞 原代培养 方法 比较 

分 类 号：R593.22[医药卫生—内科学]