Establishment And Evaluztion Of Collagen-induced Arthritis Model In Rats And Investigation Of Mechanism In CIA

OBJECTIE:To isolate and purify collagen typeâ…¡(Câ…¡) and establish the rat model of collagen-induced arthritis(CIA).To observe the physical signs, pathological and immunological changes of CIA model. In order to elucidate the mechanism of CIA.METHODS AND RESULTS:1. Preparation and analysis of pure collagen typeâ…¡from chicken sternal cartiageCâ…¡was extracted from chicken sternal cartiage by pepsin digestion in acid solvent; The contaminants including the proteoglycans and denatured and degraded protein and other collagen were extracted by guanidinium hydrochloride, removed via the salting of sodium chloride and purified through treatment with DE52 cellulose; The Câ…¡was identified by SDS-PAGE electrophoresis analysis and absorption spectrum . The results showed that the molecular size of purified Câ…¡was near to standard Câ…¡by SDS-PAGE and the absorption peak was at 226 nm. In conclusion , the method to prepare Câ…¡is simple and effective.2. Establishment and evaluztion of collagen-induced arthritis model in ratsThe rats were injected subcutaneously with Câ…¡, emulsified with complete Freund's adjuvant (CFA). The changes of secondary paw-swelling were observed and scored ; Pathological changes of synovium were examined by Hematoxylineosin (HE) stain method;antibody against collagen typeâ…¡(Câ…¡) were determined by enzyme-linked immunosorbent assay (ELISA); Interleukin-1(IL-1) and Tumor necrosis factor alpha (TNF-α) were measured by Radioimmunoassay. The results showed that the inflamm -ation and paw swelling of CIA rats increased significantly;The pathological examin -ation also revealed that pannus formed and inflammatory cells infiltrated into the synovium ;The serum antibody gainst Câ…¡, IL-1 and TNF-αwere elevated significantly. In conclusion, the collagen-induced arthritis modle in rats was established.3. Investigation of mechanism in collagen-induced arthritis in ratsConA- and LPS-induced splenocyte proliferation on CIA rats were assayed with MTT reagent ; The expression of Foxp3mRNA and TGF-β₁mRNA in the spleen were detected by reverse transcription polymerase chain reaction.The results showed that The splenic lymphocyte proliferation reaction of CIA rats were increased; Foxp3mRNA and TGF-β₁mRNA in the spleen were significantly down-regulated in model groups compared with control groups.CONCLUSIONS:1. This prepared collagen is a product of collagen of typeâ…¡, and the method is simple and effective.2. The CIA model is similar to human RA in clinical situation, immunological and pathological changes. The model could be successfully established.3. In the CIA model, changes in lymphocytes and the degree of inflammation are closely related .The function of CD4⁺CD25⁺ regulatory T cells was lowered in CIA, its lowered function was caused by the dysfunction of Foxp3,This may cause insufficiently TGF-β₁ produced, and cannot suppress proliferation of T-lymphocytes.