脐带间充质干细胞对类风湿关节炎滑膜成纤维细胞基质金属蛋白酶的影响

Effects of umbilical cord-derived mesenchymal stem cells on matrix metalloproteinases secreted by fibroblast-like synoviocytes from rheumatoid arthritis

目的探讨脐带间充质干细胞（UCMSCs）对 RA 滑膜成纤维细胞（FLSs）分泌 MMPs 的影响。方法 Wistar 大鼠制作胶原诱导关节炎（CIA）模型，第17天尾静脉输注1＆#215;106 UCMSCs，第42天处死大鼠后收集滑膜组织提取 RNA，实时荧光定量 PCR 检测滑膜组织 MMP-1、MMP-3、MMP-13的表达水平。取行膝关节置换术的 RA 和 OA 患者滑膜组织，分离培养 FLSs，比较 RA 与 OA FLSs 的 MMP-1、MMP-3和 MMP-13的表达水平。将 RA FLSs 与 UCMSCs 用 Transwell 小室共培养72 h，配对 t 检验比较共培养前后 FLSs 的 MMPs 表达水平变化及上清中 MMPs 含量，并检测 UCMSCs 在共培养过程中分泌的可溶性细胞因子水平。向培养体系中加入可溶性细胞因子的抑制剂后观察 FLSs 表达 MMPs 水平变化。2组间比较采用 t 检验，而3组及以上之间比较采用单因素方差分析。结果 CIA 大鼠滑膜组织较正常鼠高表达 MMP-1（6.9±5.4，1.3±1.4，P〈0.05）、MMP-3（6.0±6.5，1.4±1.0，P〈0.05）和 MMP-13（21.8±20.8，1.5±1.6， P〈0.05），UCMSCs 治疗后与未治疗组及成纤维细胞治疗组比较，滑膜组织 MMP-1（1.3±1.4，6.9±5.4，8.7±6.8，P〈0.05）、MMP-3（1.4±1.5，6.0±6.5，6.0±5.7，P〈0.05）和 MMP-13（3.0±3.2，22±21，22±26，P〈0.05）均表达下调。 RA 患者 FLSs MMP-1（1.8±0.9，0.9±0.7，t=2.44，P〈0.05）、MMP-3（2.6±1.7，1.1±1.0，t=2.25，P〈0.05）和MMP-13（2.4±2.3，0.6±0.7，t=2.37，P〈0.05）水平明显高于 OA 患者。经 UCMSCs 体外共培养后，MMP-13（1.3±1.2，0.9±1.2，t=3.63，P〈0.05）水平下调，而 MMP-1（1.5±1.4，6.6±6.0，t=3.90，P〈0.05）、MMP-3（7±17，22±35，t=2.86，P〈0.05）上调。共培养后 UCMSCs 表达 IDO、肝细胞生长因子（HGF）和 IL-10等可溶性细胞因子水平明显增多。加入 IL-10抗体后 UCMSCs 抑制 MMP-13的能力减弱。结论 UCMSCs 通过分泌可溶性细胞因子 IL-10抑制 RA 滑膜组织 MMPs 分泌，从而改善关节炎病情。

Objective To explore the effects of umbilical cord-derived mesenchymal stem cells （UCMSCs） on fibroblast-like synoviocytes（FLSs） from patients with rheumatoid arthritis（RA）. Methods collagen-induced arthritis（CIA） models were developed on Wistar rats and 1 ＆#215;106 UCMSCs were given by intravenous injection from tail vein on the 17th day. On day 42, rats were sacrificed and synovial tissues were obtained to detect matrix metalloproteinase （MMP）-1, MMP-3 and MMP-13. Synovial tissues from patients with RA and osteoarthritis（OA） treated by knee arthroplasty were used to isolate FLSs. RNA of FLSs were extracted to compare MMP-1, MMP-3 and MMP-13 expression. FLSs and UCMSCs were cocultured through transwell for 72 hours. Then levels of MMPs were compared in the supernatants by Luminex. The MMPs expressed by FLSs and soluble factors expressed by MSCs were detected by real time-polymerase chain reaction（PCR）. After adding antibodies to soluble factors, the MMPs expressions of RA FLSs were compared. Results MMP-1 （6.9±5.4, 1.3±1.4, P〈0.05）, MMP-3 （6.0±6.5, 1.4±1.0, P〈0.05） and MMP-13 （21.8± 20.8, 1.5±1.6, P〈0.05） expression were much higher in CIA rats compared with healthy controls. MMP-1 （1.3±1.4, 6.9±5.4,8.7±6.8, P〈0.05）, MMP-3（1.4±1.5, 6.0±6.5, 6.0±5.7, P〈0.05） and MMP-13（3.0±3.2, 〈br〉 22±21, 22±26, P〈0.05） expression were inhibited by UCMSCs in vivo. In vitro, MMP-1 （1.8±0.9, 0.9±0.7, t=2.44, P〈0.05）, MMP-3（2.6±1.7, 1.1±1.0, t=2.25, P〈0.05） and MMP-13（2.4±2.3, 0.6±0.7, t=2.37, P〈0.05） levels were higher in RA than OA FLSs. After coculture, MMP-13（1.3±1.2, 0.9±1.2, t=3.63, P〈0.05） expressed by FLSs were down-regulated, however MMP-1 （1.5±1.4, 6.6±6.0, t=3.90, P〈0.05） and MMP-3 （7±17, 22±35, t=2.86, P〈0.05） were up-regulated when analyzed by paired t-test. Soluble factors such as I-DO, HGF and IL-10 were elevated. Anti-IL-10 antibody could decrease the function of UCMSCs by inhibiting MMP-13 expression in RA FLSs. Conclusion UCMSCs ameliorates RA by secreting soluble factor IL-10, which may inhibit MMPs expressed by FLSs.