Purification, Biological Characteristics Of Chicken Type â…¡ Collagen And Interference In Rheumatoid Arthritis By Oral Tolerance

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by the chronic inflammation of the synovial membrane and destruction of articular cartilage. The pathogenesis is not completely understood. TypeⅡcollagen (CⅡ), the major constituent protein of cartilage matrix, consists of 3 identical al(Ⅱ) chains. Recent studies have shown that the development of RA is associated with CⅡ-mediated autoimmune response. Anti-natural CⅡand denatured CⅡantibodies could be detected in sera and synovial fluids of 10-30% patients with RA. Experimental arthritis may be induced in some animals immunized with CⅡ. Oral administration of CⅡfor the induction of immune tolerance can be significantly effective in the treatment of RA. But, the results of these studies vary greatly. The mechanism of anti-CⅡautoantibody formation and CⅡimmune tolerance has not be studied deeply, which overwhelmingly limit the CⅡpractical application in medicine. The main influential factors include as follow:①the CⅡsource and extraction method,②the detection conditions for CⅡantibody,③the CⅡbiological characteristics and effects of digestive enzymes to CⅡ,④the oral dosage, forms and immune state of mucous membrane in gastrointestinal tract, et al. In this study, we established a reliable method for isolating and purifying C II from chicken sternal cartilage and studied the CⅡbiological characteristics in detail. An improved indirect ELISA of anti-CⅡantibodies had be established by optimizing experimental conditions and be investigated in the RA clinical significance. In particular, we observed the enzymolysis of digestive enzymes on CⅡin vitro and in vivo explore the effects of oral CⅡon cytokine expressions of Peyer's patches (PP) and the levels of serum specific IgG, IgA, IgM in order to provide some foundation on the possibility and possible mechanism of treating rheumatoid arthritis by oral CⅡto induce immune tolerance.Ⅰ. Study on Optimizing Preparative Procedure for Isolating and Purifying TypeⅡCollagenAim:To establish a reliable method for isolating and purifying typeⅡcollagen by optimizing preparative procedure. Method:The chicken sternal cartilage was selected as raw material. Guanidine hydrochloride was used to remove the proteoglycans. The digestion manners of pepsin, sodium chloride concentrations for salting, types of DEAE anion resin were studied for extracting CⅡ. The CⅡidentification was made by SDS-PAGE.Results:It was convenient for pre-treatments of chicken sternal cartilage. The homogenate of sternal cartilage contained mainly 4 type proteins including CⅡ. The proteoglycans could be efficiently removed by 4 mol/L guanidine hydrochloride. The satisfied result was obtained by limited enzyme digestion of pepsin added by two steps, controlling the reaction temperature at 4℃and adjusting quickly the pH value to 7.5 on the supernatant after pepsin digestion. The optimizing concentration of sodium chloride for salting was 2.4mol/L. SDS-PAGE maps revealed that the bands of purified CⅡand standard CⅡwere at the same location. The relative molecular weight of a monomer was 120 kDa.Conclusion:The improved method has significant advantages of simple working process, result reliability and convenient source of raw material. It is suitable for purifying CⅡat variable scales in research works and clinic application.Ⅱ. Study on the Biological Characteristics of TypeⅡCollagenAim:To study the physicochemical characteristics of typeⅡcollagen and investigate the regular patterns of antibody production and immune reactivity.Method:The CⅡabsorption peak and isoelectric point were detected by absorption spectroscopy. The amino acid compositions were analyzed by HPLC. The specific viscosity of the CⅡsolution was measured using an Ubbelohde viscometer. The molecular weight was determined by SDS-PAGE. SDS-PAGE CⅡand Western blotting were used to analyze the immune reactivity of the CⅡand heat-denatured CⅡ(D-CⅡ). The New Zealand white rabbits were immunized with CⅡand then the rabbit sera were collected every 10 days. The improved indirect ELISA method was adopted to detect the titers of anti-CⅡantibodies.Results:SDS-PAGE showed that CⅡhad three bands(α,βandγbands), all could react with the specific anti-CⅡantibody and the relative molecular weight of a monomer was 120 kDa. D-CⅡwas pyrolyzed to more than 10 fragments showed positive in immunoblotting. The maximum absorption peak was at 230 nm and the pI was approximately 6.25. Among the amino acids identified, the contents of glycine, proline, and alanine were higher whereas that of the aromatic amino acids lower. The specific viscosity of the CⅡsolution increased in a concentration-dependent manner and decreased in a temperature-dependent manner at 25℃-45℃. When the New Zealand white rabbits were immunized with CⅡ, the antibody titers in sera detected by ELISA were 1:200 at 10 day and 1:204 800 at 20 d. After booster the antibody titers increased to 1:409 600 at 30th day and 1:819 200 at 40th day.Conclusion:The results reveal that the CⅡis a type of collagen with unique physicochemical characteristics and has higher immunogenicity with rich proline. The antiserum with high titer can be obtained when the rabbits are immunized with CⅡ.Ⅲ. Establishment of an Enzyme-linked Immunosorbent Assay on Anti-collagen TypeⅡAntibodies and Study on the Clinical SignificanceAim:To establish an enzyme-linked immunosorbent assay (ELISA) for anti-collagen typeⅡantibodies and investigate the clinical significance in RA diagnosis and treatment.Method:The chicken typeⅡcollagen, purified from sternal cartilage with above-mentioned method by us, was used as coating antigen. An improved indirect ELISA of anti-collagen typeⅡantibodies was established by optimizing experimental conditions. The serum anti-CⅡantibodies were detected in 289 RA patients and 54 normal individuals.Results:The optimized ELISA conditions for detecting serum anti-CⅡantibodies were as follow. First, CⅡstore solution (0.5 g/L) was prepared in 0.05 mol/L acetic acid. In coating, CⅡconcentration was 20μg/ml, diluted with PH7.60.05 mol/L phosphate buffer, and kept at 4℃for 12 h. In blocking step,10% goat serum was used as blocking reagent and kept at 37℃for 2 h. The sera and HRP-labelled goat anti-human IgG were respectively diluted as 1:50 and 1:2000 with PH7.40.05 mol/L tris buffer contained 10% goat serum. The dual-wavelength of 450nm/630 nm was adopt in detecting absorbance. The positive rates of serum anti--collagen typeⅡantibodies in the RA patients and normal individuals were 19.38% and 1.85% respectively. The difference was very notable between two groups (P<0.01). Conclusion:CⅡcan be used as ELISA coating antigen in detecting serum anti-CⅡantibody. The buffers containing 10% goat serum, used as blocking reagent and diluents for serum specimens and enzyme-labelled antibody, can effectively prevent nonspecific adsorption and improve the reproducibility and specificity of results. Anti-CⅡantibody can serve as a supplemental parameter in diagnosing RA patient conditions.Ⅳ. Effects of the Digestive Enzymes on CⅡin vitro and in vivoAim:To observe the enzymolysis of digestive enzymes on the native typeⅡcollage (N-CⅡ) and the denatured typeⅡcollage (D-CⅡ) in vitro and study the possible mechanism of treating RA by oral administration through supplying CⅡto mouse and rat gastrointestinal tract.Method:N-CⅡand D-CⅡwere respectively treated with pepsin, trypsin andα-chymotrypsin in vitro and the products were analyzed by SDS-PAGE and Western blotting. N-CⅡand D-CⅡwere infused to the murine stomach and duodenum. The gastric contents and intestinal contents were collected at different times and analyzed by SDS-PAGE.Results:CⅡcould be in the lyso-state in the gastrointestinal tract. After the pepsin and chymotrypsin treatments, the N-CⅡSDS-PAGE and immunoblotting graphs had no obvious change, but for D-CⅡthe positive bands in immunoblotting vanished. After the trypsin treatment,10 positive bands in immunoblotting could be seen for N-CⅡand no positive bands for D-CⅡ. N-CⅡcould not be hydrolyzed in gastric environment and could be slowly degraded in intestinal tract. D-CⅡcould be easily hydrolyzed by the digestive enzymes in the gastrointestinal tract.Conclusion:N-CⅡcan be produced active fragments by the trypsin treatment and has resistance to gastric environment, pepsin and chymotrypsin. D-CⅡcan be easily hydrolyzed by the digestive enzymes in the gastrointestinal tract. The results suggest that the inducing immunotolerance to treat RA by oral administration of CⅡis possibly concerned with the CⅡcharacteristics and trypsin enzymolysis. Ⅴ. Effects of oral typeⅡcollagen on serum antibody and the cytokine cxpression in Peyer's patchesAim:To explore the effects of oral typeⅡcollagen (CⅡ) on the morphology, cytokine expressions of Peyer's patches (PP) and the levels of serum specific IgG, IgA, IgM.Method:CⅡwas orally administrated to Kunming mice in continuous 10 days at different dosage. The CⅡor adjuvant immunization was given at 11d and 21d. The blood and Peyer's patches were collected at 11d,21d and 31d. The PP hyperplasy was observed by light microscope after HE staining. The fluorescent real time RT-PCR was used to detect the mRNA expressions of IL-17, TNF-a, IFN-y and TGF-β1 in PP lymph node. The serum specific IgG, IgA, IgM contents were detected by ELISA.Results:After oral administration of CⅡfor 10d, the PP lymph node hyperplasia was active and the cap-shape structure could be seen clearly in high dose group, the serum IgA could be detected, the gene expressions of IL-17, TNF-a and IFN-γwere inhibited. After the CⅡinitial immunity, the IgA, IgM, IL-17 levels were descended and TGF-β1 level was increased in the experiment groups as compared with control group(p<0.05 or p <0.01). After the CⅡbooster, IgA was notably increased in high dose group (p<0.05), in experiment groups IgM was still suppressed (p<0.05 or p<0.01) and TGF-β1 levels were higher than control group (p<0.05). In adjuvant immunization groups the cytokine expressions were similar to CⅡimmunization groups, the differences of serum specific IgG, IgA, IgM could not be observed as compared with control group.Conclusion:The oral administration of CⅡcan increase the serum specific IgA and suppress the gene expressions of IL-17, TNF-α, IFN-γin the Peyer's patches. It can still have inhibitory action on the serum specific IgA, IgM and IL-17 gene expressions after CⅡimmunization. The results on the changes of serum specific antibodies and cytokine gene expressions indicate that the oral CⅡwill help to induce immune tolerance, interfere in the RA development and progression and has some potential preventive and therapeutic effect.