Study On The Change Of CD4~+CD25~+ Regulatory T Cell And Its Preliminary Role In Experimental Rat Adjuvant Arthritis Model

OBJECTIVE:To analyze the change of CD4~+CD25~+regulatory T cells (CD4~+CD25~+Treg) in rat adjuvant arthritis model and to investigate their preliminary roles in the progress of arthritis. To observe the possible role of CD4~+CD25~+Treg to peritoneal macrophage, elucidate the mechanism of RA and provide certain evidence whether CD4~+CD25~+Treg could be the preventive and therapeutic target for RA.METHODS AND RESULTS:1. Establishment and evaluztion of adjuvant arthritis model in ratsHealthy male SD rats were administrated with 0.1ml complete Freud adjuvant (FCA) (10g?L-1) via toe endermic injection. The change of secondary paw-swelling was observed and scored; Animals were sacrificed at every time point, Pathological changes of synovium were examined by Hematoxylin eosin (HE) stain method, then the samples of celiac lymphocyte and peritoneal macrophage were collected. The proportion of CD4~+CD25~+Treg was detected by flow cytometry. The results showed that the inflammation and paw swelling of AA rats increased significantly; The pathological examination also revealed that inflammatory cells infiltrated into the synovium, The synoviocytes were hyperplasia and collagen was exuded to form cellulose deposition. In conclusion, adjuvant arthritis model in rats was established successfully. 2. Change and its significance of CD4~+CD25~+ regulatory T cells from celiac lymph node and peripheral blood in experimental rat adjuvant arthritis modelOn the basis of successful rat adjuvant arthritis model, collectted celiac lymph nodes and peripheral blood at the different stage of AA, which were 1d, 3d, 10d, 17d, 21d, 24d and 28d, prepare lymphocyte supernants according to the relative literature; ConA- and LPS-induced splenocyte proliferation on AA rats were assayed with MTT reagent; the proportion of CD4~+CD25~+Treg as well as those stimulated with ConA for 48h was detected by flow cytometry; The relationship between CD4~+CD25~+Treg and arthritis symptom was processed by Pearson correlation analysis. The results showed that the proportion of CD4~+CD25~+Treg in AA group was significantly higher than control group at 1d, 24d and 28d; The results showed that the lymphocyte proliferation reaction of AA rats were lowered; The proportion of CD4~+CD25~+Treg in control group was significantly increased after stimulated with ConA for 48h, while the difference of AA group had no significance; There was positive correlation between CD4~+CD25~+Treg and arthritis symptom.3. Study of the preliminary role of CD4~+CD25~+Treg in AACeliac LC and PMΦwere isolated according to literature, LC was co-cultured with PMΦfor 48 hours and the cultivation supernant was collected for detecting TNF-αand IL-6 which was measured by Radioimmunoassay. The level of TNF-αand IL-6 in co-culture supernants of normal LC and AA PMΦwere slightly lower than AA PMΦ, but obviously higher than normal PMΦ, while those in co-culture supernants of AA LC and normal PMΦdecreased significantly. This suggested that CD4~+CD25~+Treg from normal LC presented a negative feedback effect on AA PMΦ, and CD4~+CD25~+Treg from AA LC exerted obviously inhibitive effect on the function of normal PMΦ.CONCLUSIONS:1. This study discovered the rule of the dynamic change of the proportion of CD4~+CD25~+ Treg from celiac lymph node and peripheral blood at different stages of AA, and the changes of CD4~+CD25~+Treg and the degree of inflammation were positively correlated.2. CD4~+CD25~+Treg may play the role in inhibting PMΦsecreting TNF-αand IL-6 in the co-culture system in vitro.