人类风湿关节炎滑膜成纤维细胞-软骨-重度联合免疫缺陷鼠模型的研制

Study on the novel models with human rheumatoid arthritis synovial fibroblasts-cartilage-SCID micefor rheumatoid arthritis

目的建立人类风湿关节炎（RA）滑膜成纤维细胞（SFs）．软骨．重度联合免疫缺陷（SCID）小鼠模型，以骨关节炎滑膜成纤维细胞（OASFs）作对照，探讨RASFs在RA发病中的作用及机制。方法将培养传代至第4代的RASFs／OASFs经5-澳脱氧尿嘧啶核苷体（5-Brdu）标记后注入可吸收明胶海绵，与人的正常软骨一起移植入SCID小鼠背部皮下，第30天处死模型，剪取移植物和鼠双膝关节作组织学观察，免疫组织化学检测5-Brdu和Vimehtin阳性细胞，酶联免疫吸附试验（ELISA）检测血清中人基质金属蛋白酶（MMP）-3和人白细胞介素（IL）-6含量。统计学方法采用两独立样本秩和检验。结果①2组血清中均可检测出人MMP-3，仅在RASFs组检测到1例IL-6。②RASFs组移植的软骨侵蚀程度（0-6±0．7与O．3±0．5）和软骨降解程度（2．3±0．8与1．7±1．0）较OASFs组有增高趋势，但差异均无统计学意义（P〉0．05）。③RASFs组鼠膝关节的滑膜增生程度（3．1±0．8与1．7±1．0，P〈O．01）和软骨侵蚀程度（1-6±1．7与0-6±1．4，P〈O．05）均显著高于OASFs组。④在SCID鼠皮下、移植软骨处、鼠膝关节滑膜及骨髓中检测出5-Brdu及Vimentin阳性的SFs。结论传代培养的RASFs在SCID鼠皮下存活并保持其侵袭性生长的特点，且能迁移至远处关节中诱发关节炎症。

Objective To establish the models with human rheumatoid arthritis （RA） synovial fibroblasts （RASFs）-cartilage-severe combined immune deficient （SCID） mice for the study of the RASFs in the pathogenesis of RA. Methods The 4th passage RASFs were marked with 5-bromodexyuridine（5-Brdu） and injected into a cavity of inert sterile gel sponge, then with the normal human cartilage co-implanted in the back subcutaneously of SCID mice to set up a novel model of RA. Osteoarthritis synovial fibroblasts （OASFs） were injected as control group. Thirty days after She surgery, the mice were killed, the grafts and the knee joints were proceed with histological study and immunohistochemistry to detect the expression of 5-Brdu and Vimentin in synoviocytes. The serum level of interleukin （IL）-6 and matrix metalloproteinase （MMP）-3 were detected with enzyme-linked immunosorbent assay （ELISA）. Results Twenty-three mice survived except for one mouse in the RASFs group died of anesthesia. ① Only in one case in the RASFs group, the IL-6 was detected, the others were unable to be detected. The MMP-3 in the OASFs group was （40＋17） pg/ml, but in RASFs group only one case was detected.② There were 4 and 3 implanted cartilages loss in the RASFs group and OASFs group respectively. The histological scores of cartilage invasion by synoviocytes and cartilage degradation in grafts were higher in RASFs groups than in OASFs groups （0.6±0.7 vs 0.3±0.5, 2.3±0.8 vs 1.7±1.0 respectively）. ③ The histological scores of synovial hyperplasia and cartilage invasion in the knee joints was significantly higher in the RASFs group than in the OASFs group （3.1±0.8 vs 1.7±1.0, P〈O.O1,1.6＋1.7 vs 0.6：t：1.4, P〈0.05 respectively）. ④ During the grafts, a lot of 5-Brdu and Vimentin markers positive synovio-cytes were found in the mice subcutaneous tissue, but manipulus positive synoviocytes were found on the area of cartilage invasion in both groups. In knee joints, single positive synoviocytes could be detected in bone marrow and hyperplasic area of the synovial tissue in both groups. Conclusion The isolated RASFs can survival and have the ability to invade and degrade the cartilage in vivo without the limitation of immunity induced inflammations, and can also migrate to the synovial joints in distance and induce arthritis.