来源：风湿病与关节炎,2021,10（1）:1-5,15.

类风湿关节炎滑膜成纤维细胞增殖及巨自噬的表达水平

（基础研究）

邵　鑫¹，蒋先虹¹，王　瑞¹，蒲强红¹，刘　福²

　　【摘　要】目的：探讨体外原代培养的类风湿关节炎滑膜成纤维细胞（RA-FLS）中是否存在巨自噬现象以及在类风湿关节炎（RA）中的发病机制。方法：培养原代RA-FLS，以骨关节炎（OA）患者膝关节滑膜组织中培养的骨关节炎滑膜成纤维细胞（OA-FLS）作为对照组。采用MTT法检测细胞增殖活力；流式细胞仪检测细胞凋亡；采用实时荧光定量聚合酶链反应（RT-qPCR）、蛋白免疫印迹法（Western Blot）分别检测2组细胞中巨自噬标志性因子LC3、P62 mRNA及蛋白的相对表达水平，激光共聚焦显微镜检测LC3、P62蛋白的表达。结果：从患者膝关节滑膜组织中成功分离、培养出RA-FLS与OA-FLS，MTT结果显示，RA-FLS增殖活力明显强于OA-FLS；流式细胞仪结果显示，RA-FLS的细胞凋亡减少；RT-qPCR、Western Blot结果表明，RA-FLS与OA-FLS相比，LC3 mRNA及蛋白的相对表达水平升高、P62 mRNA及蛋白的相对表达水平降低，差异均有统计学意义（P < 0.05）；激光共聚焦显微镜结果显示，RA-FLS中LC3表达更高，P62表达更低。结论：RA-FLS中LC3 mRNA及蛋白的相对表达水平明显增高，P62 mRNA及蛋白的相对表达水平显著降低，提示RA中巨自噬水平活化，这可能是导致RA-FLS出现凋亡抵抗，增殖活性明显高于OA-FLS的潜在原因。

　　【关键词】　关节炎，类风湿；骨关节炎；滑膜成纤维细胞；巨自噬；LC3；P62

Proliferation and Expression of Macroautophagy in Synovial Fibroblasts of Rheumatoid Arthritis

SHAO Xin,JIANG Xian-hong,WANG Rui,PU Qiang-hong,LIU Fu

　　【ABSTRACT】Objective:To investigate the existence of macroautophagy in rheumatoid arthritis synovial fibroblasts（RA-FLS）in vitro and its pathogenesis.Methods:Primary RA-FLS were cultured,and OA-FLS were used as a control group.MTT was used to detect cell proliferation activity;flow cytometry was used to detect cell apoptosis;RT-qPCR and Western Blot were used to detect the relative expression levels of LC3,P62 mRNA and protein in the two groups;and laser confocal microscopy was used to detect the expression levels of LC3 and P62.Results:RA-FLS and OA-FLS were successfully isolated and cultured from synovial tissue of knee joint.MTT results showed that the proliferation activity of RA-FLS was significantly stronger than that of OA-FLS.Flow cytometry results showed that the apoptosis of RA-FLS was reduced;RT-qPCR and Western Blot results showed that compared with OA-FLS,the relative expression levels of LC3 mRNA and protein of RA-FLS were higher,and P62 protein of RA-FLS was lower.The difference was statistically significant（P < 0.05）.Laser confocal microscopy showed that LC3 expression was higher and P62 expression was lower in RA-FLS.Conclusion:The relative expression levels of LC3 mRNA and protein in RA-FLS were significantly increased,while the relative expression levels of P62 mRNA and protein in RA-FLS were significantly decreased,suggesting that the level of macroautophagy was activated in RA,which may be the potential reason for the apoptosis resistance and proliferation activity of RA-FLS being significantly higher than those of OA-FLS.

　　【Keywords】 arthritis,rheumatoid;osteoarthritis;synovial fibroblasts;macroautophagy;

LC3;P62