猕猴肺成纤维细胞的不同分离培养方法及鉴定比较  

Comparison of different isolation and culture methods and identification of rhesus monkey lung fibroblasts

作　　者：许振 蒋海峰 张磊[1] 刘潇一 董婷玉 檀学文 严尚学[1] 常艳[1] 魏伟[1] Xu Zhen;Jiang Haifeng;Zhang Lei;Liu Xiaoyi;Dong Tingyu;Tan Xuewen;Yan Shangxue;Chang Yan;Wei Wei(Institute of Clinical Pharmacology,Anhui Medical University,Key Lab of Anti-inflammatory and Immune Medicine,Ministry of Education,Anhui Collaborative Innovation Center of Anhui-inflammatory and Immune Medicine,Hefei 230032)

机构地区：[1]安徽医科大学临床药理研究所,合肥230032

出　　处：《安徽医科大学学报》2022年第7期1041-1047,共7页Acta Universitatis Medicinalis Anhui

基　　金：安徽高校重点科研平台协同创新项目(编号:GXXT-2020-065)。

摘　　要：目的比较不同方法提取猕猴肺成纤维细胞的效率及对细胞相关功能的影响,为获得类似人肺成纤维细胞提供有效方法。方法采用两种猕猴肺成纤维细胞的提取方法,组织块黏附法和胶原酶联合消化+组织黏附法。倒置显微镜观察细胞形态,免疫荧光鉴定猕猴肺成纤维细胞,CCK-8检测细胞活力,流式细胞仪检测肺成纤维细胞标志物α-SMA的表达,凋亡试剂盒检测细胞凋亡,Western blot检测细胞α-SMA蛋白水平表达。结果组织黏附法贴壁的组织块72 h后可见小而亮的细胞爬出,4~5 d后细胞小范围爬出,呈长梭形;7 d后可见细胞大范围爬出,形成单层细胞,传代后细胞均呈长梭形。用胶原酶联合消化+组织黏附法处理后24 h即可见小而亮的细胞从组织块中爬出,48 h后可见细胞大范围爬出,细胞呈长梭形;4~5 d后可形成单层细胞,传代后的细胞均呈长梭形,用免疫荧光鉴定均表达α-SMA。实验结果显示胶原酶联合消化+组织黏附法提取的肺成纤维细胞的活力明显高于组织黏附法所得细胞活力。TGF-β1刺激肺成纤维细胞后,胶原酶联合消化+组织黏附法提取的细胞增殖更快,α-SMA蛋白表达更高。结论两种方法均能体外分离出猕猴肺成纤维细胞,胶原酶消化法提取猕猴肺成纤维细胞所需的时间较短且状态较好,TGF-β1刺激后相关蛋白水平表达更稳定,是获得猕猴肺成纤维细胞的有效方法,也是获得接近人原代肺成纤维细胞的有效方法。Objective To compare the efficiency of different methods for extracting rhesus monkey lung fibroblasts and their effects on functions, so as to provide a method for obtaining primary lung fibroblasts that are closer to human fibroblasts. Methods Two extraction methods for rhesus monkey lung fibroblasts were used, direct tissue block adhesion method and collagenase combined digestion with tissue block adhesion method. The cell morphology was observed with the inverted microscope, the purity of isolated rhesus monkey lung fibroblasts was identified by immunofluorescence, cell viability was detected by CCK-8, the expression of α-SMA was detected by flow cytometry and the effect of long-term in vitro culture on cell apoptosis was detected by apoptosis kit. Western blot was used to detect the expression of α-SMA protein. Results The combined digestion with collagenase and tissue block adhesion method could see small and bright cells crawling out in 24 hours, and cells could be seen crawling out in a large area after 48 hours. The cells were in a long spindle shape, after 4 days to 5 days, a single layer of cells could be formed. Identified by immunofluorescence, all cells expressed α-SMA. Tissue adhesion method showed small and bright cells crawling out after 72 hours. After 4 days to 5 days, the cells crawled out in a small area and showed a long spindle shape. After a week, the cells crawled out in a large area and formed a single layer of cells and the cells are all expressed α-SMA by immunofluorescence. The experimental results showed that the cell viability of the cells crawled out by the collagenase digestion method was significantly higher than that of the tissue adhesion method. After TGF-β1 stimulates the cells, the cells extracted by collagenase digestion method proliferated faster and expressed α-SMA more obviously. Conclusion Both methods can isolate rhesus monkey lung fibroblasts in vitro, but the collagenase digestion method extracts cells in a shorter time and in better condition. The expression o

关 键 词：猕猴 肺 原代培养 肺成纤维细胞 

分 类 号：R563.9[医药卫生—呼吸系统]