邵鑫　蒋先虹　王瑞　蒲强红　刘福

【摘 要】目的：探討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

类风湿关节炎（rheumatoid arthritis，RA）主要病理特征为滑膜增生、血管翳形成及软骨破坏，最终可致残疾和死亡。国内调查结果显示，RA等关节疾病和脑血管病是我国两大主要致残原因[1]。近年来，随着对本病认识的加深和新治疗方法的出现，其预后得到明显的改善，但总预后仍不容乐观，RA的发病机制至今仍未完全明确[2]。巨自噬过程在自身免疫性疾病中的作用逐渐成为研究热点，大量研究表明巨自噬在RA发病机制、病程进展中占有关键性地位[3]。类风湿关节炎滑膜成纤维细胞（RA-FLS）是一种以肿瘤样方式异常增殖的细胞，细胞活性增强，凋亡减少，导致滑膜异常增生，这在RA的发病机理中起关键作用[4]。……