CCL18通过Cdc42-GTP酶促进类风湿关节炎成纤维样滑膜细胞迁移、侵袭破坏关节的作用

Migration and invasion of fibroblast-like synoviocytes (FLS) which cannot be intervened by present therapies lead to persistent joint destruction and disability in rheumatoid arthritis (RA). Some refractory RA patients didn’t reach remission and kept persistent joint destruction even though progressive therapies. Our pre-experiments showed chemokine CCL18 level significantly increased in joints of refractory RA patients; and CCL18 in vitro promoted F-actin polymerization, cellular migration/invasion and production of matrix metalloproteinases which mediated cartilage degradation in RA-FLS. By expression profile microarray chip, we found CCL18 upregulated the transcription of cell division cycle protein 42 (Cdc42) in RA-FLS, which has been verified. It has been known that Cdc42-GTPase promotes cellular migration/invasion through acting on cytoskeleton. To sum up, we propose a hypothesis that CCL18 promote the migration/ invasion of RA-FLS through Cdc42 GTPase. We will perform in vitro gene regulation, in vivo animals experiments, automatic cell array scan with high content screening to analyze cytoskeleton and real time cellular analysis for dynamic curve of cell migration/invasion, in order to illustrate the promoting role and mechanism of CCL18 on migration and invasion of RA-FLS, for therapeutic target to retard joint destruction in RA.

类风湿关节炎(RA)成纤维样滑膜细胞(FLS)迁移/侵袭导致关节破坏及患者残疾，现有药物不能阻断此过程，部分患者规律治疗后，病情不缓解或持续关节破坏，属难治性RA。我们预实验发现难治性RA关节局部趋化因子CCL18显著升高，且CCL18在体外能促进RA-FLS的F-肌动蛋白聚合、迁移、侵袭及产生基质金属蛋白酶3(与侵袭软骨相关)，表达谱芯片发现CCL18显著上调RA-FLS中细胞分裂周期蛋白(Cdc)42转录并经验证。目前已知Cdc42-GTP酶通过影响细胞骨架而驱动细胞迁移/侵袭。故我们提出科学假说：CCL18通过激活Cdc42-GTP酶促进RA-FLS迁移、侵袭破坏关节。本项目拟通过体外基因调控及体内动物实验，结合全自动细胞组高内涵定量分析细胞骨架、实时细胞动态分析等技术，阐明CCL18促进RA-FLS迁移及侵袭破坏关节的作用及可能机制，为寻找控制RA关节破坏治疗靶点提供新的理论依据。

类风湿关节炎（RA）成纤维样滑膜细胞（FLS）迁移与侵袭导致持续关节破坏，但现有治疗不能阻断此过程。关节滑膜衬里层的FLS位于表面，紧密排列，细胞间基本无间质填充，近似上皮细胞。正常情况下，仅一层细胞排列。当发生RA时，RA-FLS大量增殖伴凋亡减少、抛锚处独立生长及缺乏接触抑制，衬里层细胞可增至超过6层，缺乏基底膜与衬里下层细胞分隔。RA-FLS还具有迁移侵袭等间质细胞特性，故类似于上皮细胞来源肿瘤的上皮间质转化（EMT）。本研究首次报道RA患者关节滑液具有高浓度的CCL18，并与RA关节破坏进展相关；CCL18中和抗体可拮抗RA关节滑液诱导的RA-FLS迁移；Oris迁移试验及Transwell实验等提示重组CCL18在体外促进RA-FLS及正常FLS迁移侵袭。因此，研发针对CCL18的中和抗体用于关节局部治疗，有望延缓RA关节破坏进展。下一步旨在开发针对CCL18的中和抗体（单抗或者纳米抗体），并在胶原诱导关节炎的动物模型中进行关节腔注射，探讨能否减轻关节肿痛及关节破坏，以期获得更多临床前数据，评估能否用于临床试验。