Tetramerized single-chain variable fragment (ScFv) of anti-cyclic citrullinated peptide (TeAb-CCP) is a constructed tetramerized ScFv of anti-cyclic citrullinated peptide (CCP) antibodies with p53 tetrameric domain, aim to investigate its effect on fibroblast-like synoviocytes (FLSs) proliferation, migration, invasion, and production of inflammatory mediators in the in vitro co-culture system of peripheral mononuclear cells (PBMCs) and FLSs. TeAb-CCP was constructed by modifying a monovalent ScFv antibody to CCP with p53 tetrameric domain to improve its affinity. FLSs were isolated and cultured from rheumatoid arthritis (RA) patients and control subjects. A co-culture system of peripheral mononuclear cells (PBMCs) and FLSs was used. FLSs proliferation, migration, and invasion were measured by MTT, scratch test, and Transwell chamber. Supernatants were measured for cytokines, chemokines, metalloproteinases, and anti-CCP antibodies by Luminex liquid phase protein chip and ELISA. TeAb-CCP significantly inhibited FLSs proliferation in a dose-dependent mode, with maximal action at concentration of 100 μg/ml on the 7th day in the co-culture system with PBMCs and FLSs, but not the same with only FLSs. TeAb-CCP significantly suppressed FLSs migration and invasive ability compared with the controls. Significantly lower levels of interleukin (IL)-6, IL-8, RANKL, protein arginine deiminase (PAD)-2, PAD4, metalloproteinase (MMP)-1 and MMP-3 and anti-CCP antibodies were found in co-culture supernatant of TeAb-CCP group. In contrast, transforming growth factor-β (TGF-β) and tissue inhibitor of metalloproteinases-2 (TIMP-2) was significantly increased in the TeAb-CCP group. No significant difference of IL-1a, IL-10, IL-17, TNFα, VEGF, and FGF was found between two groups. As a blocking antibody, TeAb-CCP can significantly inhibit PBMCs of RA to produce pro-inflammatory mediators, and furthermore, inhibit the proliferation, activation, migration, and invasion of FLSs in vitro. In turn, it is suggested that citrullinated modified self-epitopes may be a new target for RA therapy.

抗环瓜氨酸肽 (TeAb-CCP) 四聚化单链可变片段 (ScFv) 是构建的具有 p53 四聚体结构域的抗环瓜氨酸肽 (CCP) 抗体的四聚化 ScFv，旨在研究其对成纤维细胞样滑膜细胞的影响(FLS)体外炎症介质的增殖、迁移、侵袭和产生外周单核细胞 (PBMCs) 和 FLSs 的共培养系统。TeAb-CCP 是通过用 p53 四聚体结构域修饰针对 CCP 的单价 ScFv 抗体来构建的，以提高其亲和力。从类风湿性关节炎 (RA) 患者和对照受试者中分离和培养 FLS。使用了外周单核细胞 (PBMC) 和 FLS 的共培养系统。通过MTT、划痕试验和Transwell小室测量FLSs增殖、迁移和侵袭。通过 Luminex 液相蛋白芯片和 ELISA 测量上清液中的细胞因子、趋化因子、金属蛋白酶和抗 CCP 抗体。TeAb-CCP 以剂量依赖性方式显着抑制 FLSs 增殖，在与 PBMCs 和 FLSs 共培养系统中的第 7 天，最大作用浓度为 100 μg/ml，但仅与 FLSs 不同。与对照组相比，TeAb-CCP 显着抑制了 FLSs 迁移和侵袭能力。在共培养上清液中发现显着较低水平的白细胞介素 (IL)-6、IL-8、RANKL、蛋白精氨酸脱亚氨酶 (PAD)-2、PAD4、金属蛋白酶 (MMP)-1 和 MMP-3 以及抗 CCP 抗体TeAb-CCP 组。相反，在 TeAb-CCP 组中，转化生长因子-β (TGF-β) 和金属蛋白酶组织抑制剂-2 (TIMP-2) 显着增加。两组间IL-1a、IL-10、IL-17、TNFα、VEGF、FGF无显着差异。作为阻断抗体，TeAb-CCP可以显着抑制RA的PBMCs产生促炎介质，进而抑制FLSs的增殖、活化、迁移和侵袭。在共培养上清液中发现显着较低水平的白细胞介素 (IL)-6、IL-8、RANKL、蛋白精氨酸脱亚氨酶 (PAD)-2、PAD4、金属蛋白酶 (MMP)-1 和 MMP-3 以及抗 CCP 抗体TeAb-CCP 组。相反，在 TeAb-CCP 组中，转化生长因子-β (TGF-β) 和金属蛋白酶组织抑制剂-2 (TIMP-2) 显着增加。两组间IL-1a、IL-10、IL-17、TNFα、VEGF、FGF无显着差异。作为阻断抗体，TeAb-CCP可以显着抑制RA的PBMCs产生促炎介质，进而抑制FLSs的增殖、活化、迁移和侵袭。在共培养上清液中发现显着较低水平的白细胞介素 (IL)-6、IL-8、RANKL、蛋白精氨酸脱亚氨酶 (PAD)-2、PAD4、金属蛋白酶 (MMP)-1 和 MMP-3 以及抗 CCP 抗体TeAb-CCP 组。相反，在 TeAb-CCP 组中，转化生长因子-β (TGF-β) 和金属蛋白酶组织抑制剂-2 (TIMP-2) 显着增加。两组间IL-1a、IL-10、IL-17、TNFα、VEGF、FGF无显着差异。作为阻断抗体，TeAb-CCP可以显着抑制RA的PBMCs产生促炎介质，进而抑制FLSs的增殖、活化、迁移和侵袭。TeAb-CCP组共培养上清液中发现金属蛋白酶(MMP)-1、MMP-3和抗CCP抗体。相反，在 TeAb-CCP 组中，转化生长因子-β (TGF-β) 和金属蛋白酶组织抑制剂-2 (TIMP-2) 显着增加。两组间IL-1a、IL-10、IL-17、TNFα、VEGF、FGF无显着差异。作为阻断抗体，TeAb-CCP可以显着抑制RA的PBMCs产生促炎介质，进而抑制FLSs的增殖、活化、迁移和侵袭。TeAb-CCP组共培养上清液中发现金属蛋白酶(MMP)-1、MMP-3和抗CCP抗体。相反，在 TeAb-CCP 组中，转化生长因子-β (TGF-β) 和金属蛋白酶组织抑制剂-2 (TIMP-2) 显着增加。两组间IL-1a、IL-10、IL-17、TNFα、VEGF、FGF无显着差异。作为阻断抗体，TeAb-CCP可以显着抑制RA的PBMCs产生促炎介质，进而抑制FLSs的增殖、活化、迁移和侵袭。体外。反过来，这表明瓜氨酸修饰的自身表位可能是 RA 治疗的新靶点。