钙网织蛋白促进新生血管形成在类风湿关节炎发病机制中的作用

摘要

目的：探讨钙网织蛋白（CRT）促进新生血管形成在类风湿关节炎（RA）发病机制中的作用。方法采用酶联免疫吸附试验（ELISA）法检测活动期RA患者、骨性关节炎（OA）患者和健康对照组（HC）血清以及RA和OA患者关节滑膜液中CRT的含量。采用免疫组织化学（IHC）法检测CRT在RA和OA患者关节滑膜组织中的表达。应用人脐静脉内皮细胞（HUVECs）体外模型，分别采用MTT实验、细胞划痕实验及构建体外血管生成的三维模型检测CRT在HUVECs增殖、迁移及管腔形成中的作用。结果 RA组血清中CRT含量[（6.4±3.1）µg/L]明显高于OA组[（3.7±0.9）µg/L]及HC组[（3.4±1.0）µg/L]；RA组滑膜液中CRT含量[（6.9±3.4）µg/L]明显高于OA组[（3.9±0.7）µg/L]，差异有统计学意义（P<0.01）。CRT在RA滑膜组织中高表达，主要位于滑膜衬里层和衬里下层的血管内皮细胞及血管周围、炎性细胞内外等，而OA滑膜组织中CRT仅见极少量的表达。MTT实验、细胞划痕实验及体外血管生成三维模型检测结果显示，CRT具有明显促进HUVECs增殖、迁移及管腔形成的作用。结论 CRT在RA患者血清、滑膜液及滑膜组织中表达均升高，CRT可能通过促进新生血管形成参与RA滑膜炎和血管翳形成的发病机制。%Objective Calreticulin (CRT)is a multifunctional protein of endoplasmic reticulum implicated in the pathogenesis of rheumatoid arthritis(RA). The present study was undertaken to determine whether CRT was involved in an⁃giogenesis events in RA. Methods Serum CRT levels were measured by enzyme-linked immnuosorbent assay(ELISA)in 106 patients with established RA, 75 osteoarthritis(OA)and 80 healthy controls(HC). CRT levels in synovial fluid were al⁃so measured in 25 RA and 22 OA patients. The expression of CRT in synovial tissue was examined by immunohistology. In order to investigate the role of CRT on angiogenesis, human umbilical vein endothelial cells(HUVECs)were isolated and cultured for in vitro experiments. The proliferation, migration and tube formation of HUVECs following CRT stimulation were examined in vitro by MTT assay, scratch wound healing assay and tube formation assay. Results Our results showed a sig⁃nificantly higher concentration of CRT in serum [(6.4±3.1) µg/L] of RA patients compared to that of OA [(3.7±0.9) µg/L, P<0.01] and HC [(3.4±1.0) µg/L, P<0.01];and significantly higher CRT in synovial fluid [(6.9±3.4) µg/L] of RA vs OA [(3.9± 0.7) µg/L, P<0.01]. Increased CRT expression predominantly localized to vascular endothelial cells, inflammatory cells and perivascular areas in both the lining and sublining layers of RA synovial tissue. Furthermore, CRT significantly promoted the proliferation, migration and tube formation of HUVECs, as showed by MTT assay, scratch wound healing assay and tube for⁃mation assay. Conclusion These findings suggested that CRT may be involved in synovitis and pannus formation events via promoting angiogenesis in RA.