LYVE1+巨噬细胞在RA患者关节滑膜组织中表达变化及对RA-FLS细胞迁移、侵袭、FMT的抑制作用

Expression changes of LYVE1+macrophages in synovial tissues of joints in patients with RA and their inhibitory effects on migration,invasion,and FMT

目的观察淋巴管内皮受体-1(LYVE1)+巨噬细胞在类风湿性关节炎(RA)患者关节滑膜组织中的表达变化及对RA成纤维样滑膜细胞(RA-FLS)迁移、侵袭、向肌成纤维细胞转化(FMT)的抑制作用。方法采用免疫荧光染色法对45例RA患者及45例骨关节炎(OA)患者滑膜组织LYVE1、CD68进行定性、定量检测。取对数生长期人单核白血病细胞THP-1,在培养液中加入LYVE1过表达慢病毒,培养48 h获得表达LYVE1的THP-1细胞,在表达LYVE1的THP-1细胞中加入100 ng/mL的佛波酯(PMA)诱导培养48 h,获得LYVE1+巨噬细胞;另取部分THP-1细胞,仅加入100 ng/mL的PMA诱导培养48 h获得LYVE1-巨噬细胞。取对数生长期人类风湿性关节炎成纤维细胞MH7A分为LYVE1+巨噬细胞组、LYVE1-巨噬细胞组,分别加入LYVE1+巨噬细胞、LYVE1-巨噬细胞,另将仅含培养基的小室设为空白对照组,采用划痕实验观察各组细胞的迁移能力。取MH7A细胞分为A组、B组,分别加入LYVE1+巨噬细胞、LYVE1-巨噬细胞,将仅含培养基小室设为C组,采用Transwell侵袭实验观察各组细胞的侵袭能力。取MH7A细胞分为一组、二组,分别加入LYVE1+巨噬细胞、LYVE1-巨噬细胞,将仅含培养基小室设为空白组,培养48 h时采用实时定量PCR法检测各组MH7A细胞FMT相关基因(COL1A1、fibronectin、α-SMA)的mRNA。结果RA与OA患者滑膜组织中LYVE1、CD68表达位置基本重叠;RA与OA患者滑膜组织LYVE1相对表达量分别为0.319±0.033、1.000±0.159,二者比较,P<0.05。与LYVE1-巨噬细胞组、空白对照组比较,培养24、48 h时LYVE1+巨噬细胞组细胞划痕愈合比低(P均<0.05);与B组、C组比较,培养24 h时A组细胞穿膜细胞数少(P均<0.05);与二组、空白组比较,培养48 h时一组细胞COL1A1 mRNA、fibronectin mRNA、α-SMA mRNA相对表达量少(P均<0.05)。结论RA患者关节滑膜组织中LYVE1+巨噬细胞低表达。LYVE1+巨噬细胞可抑制RA-FLS的迁移、侵袭及FMT。

Objective To observe the expression changes of lymphatic vessel endothelial receptor-1(LYVE1)+mac‐rophages in the synovial tissues of patients with rheumatoid arthritis(RA)and their inhibitory effects on migration,inva‐sion,and fibroblast-to-myofibroblast transformation(FMT).Methods Immunofluorescence staining was used to qualita‐tively and quantitatively detect LYVE1 and CD68 in synovial tissues of 45 RA patients and 45 osteoarthritis(OA)pa‐tients.Human monocytic leukemia THP-1 cells in the logarithmic growth phase were treated with LYVE1 overexpression lentivirus in the culture medium,and LYVE1-expressing THP-1 cells were obtained after 48 h of cultivation.These cells were induced with 100 ng/mL phorbol 12-myristate 13-acetate(PMA)for 48 h to obtain LYVE1+macrophages.Another portion of THP-1 cells were induced by only 100 ng/mL PMA for 48 h to obtain LYVE1-macrophages.Human rheumatoid arthritis fibroblasts MH7A in the logarithmic growth phase were divided into the LYVE1+-macrophage group and LYVE1 macrophage group,which were added with LYVE1+-macrophages and LYVE1 macrophages,separately.In addition,the cells containing only medium were set as the blank control group.Scratch experiment was performed to observe the migra‐tion ability of cells in each group.MH7A cells were divided into groups A,B,and C,which were added with LYVE1+-macrophages,LYVE1 macrophages,and only culture medium,respectively.Transwell invasion experiment was conduct‐ed to observe the invasion ability of cells in each group.MH7A cells were divided into the group one,group two,and the blank control group,which were added with LYVE1+-macrophages,LYVE1 macrophages,and only culture medium,re‐spectively.Real-time quantitative PCR was used to detect the mRNA expression of FMT-related genes(COL1A1,fibro‐nectin,α-SMA)in MH7A cells after 48 h of culture.Results The expression locations of LYVE1 and CD68 in the syno‐vial tissues of RA and OA patients overlapped.The relative expression levels of LYVE1 in the synovial tissues of RA and OA patients were 0.319±0.033 and 1.000±0.159,respectively,with statistically significant difference between these two-groups(both P<0.05).Compared with the LYVE1 macrophage group and the blank control group,the LYVE1+macro‐phage group had lower cell scratch healing at 24 and 48 h of cultivation(all P<0.05).Compared with groups B and C,the number of transmembrane cells in the group A was smaller at 24 h of cultivation(P<0.05).Compared with groups two and the blank control group,the relative expression levels of COL1A1 mRNA,fibronectin mRNA,andα-SMA mRNA in the group one were lower at 48 h of cultivation(all P<0.05).Conclusion LYVE1+macrophages are underexpressed in the synovial tissues of RA patients,and LYVE1+macrophages can inhibit the migration,invasion,and FMT.