Study On The Biochemicstry And Immune Tolerance Of Collagen And Collagen Peptide From Blue Shark

The oral tolerance(OT)stimulates small intestinal mucosa especially the intestine-related normal lymphoid immune function to block or inhibit autoimmunity.In other words,autoimmune diseases are restrained by oral exogenous protein similar to the target tissue.So far,there have been many researches on this treatment of autoimmune diseases as treatment of multi-skin sclerosis with bovine myelin,treatment of Rheumatoid Arthritis(RA)with TypeⅡCollagen(CⅡ),treatment of retinitis with Bovine S antigen,treatment of encephalomyelitis with alkaline myelin,treatment of Type 1 diabetes mellitus with human insulin,etc.Among them,the treatment of Rheumatoid Arthritis(RA)by oral TypeⅡCollagen(CⅡ)has already gone into the clinical research with remarkable efficacy and safety.Oral antigen induces OT in a dose-dependent manner,which is also related with the health of the immune system、types and nature of the antigen,etc.With uncertain mechanism remaining to be studied.There have been many researches about CⅡ and RA to explore the pathogenesis of RA also to develop the novel drugs for the treatment of RA both in domestic and abroad.However,there remain some problems about OT treatment of RA like the selection of RA like the selection of the antigen,the control of dosage of CⅡ antigen,by which more effective results can be achieved.Meanwhile,individual difference of immune response with the same dosage of CⅡ among patients should be considered.In addition,most of the studies nowadays use animal models instead of human clinical trials,which makes the effectiveness of the treatment less convincing.So,as a potential treatment of RA,OT treatment has the underlying research value.The effect of CⅡ to arthritis by inducing immune tolerance also with its relationship with the gut lymphoid immune system have been proved.But the function of other parts of lymphoid immune system and the molecular mechanism of the antigen receptor-binding of CⅡ on the immune cells’ surface in its initial phase remain unknown.Therefore,it has much scientific significance and application value to study on the molecular mechanism of the relationship between OT and both the molecular size and structure of CⅡ.In this paper,the OT treatment of RA which is induced by TypeⅡ Collagen of Shark(SCⅡ)from blue shark(Prionace glauca)cartilage and the TypeⅡ Collagen peptide of Shark(SCⅡ-P)through further enzymolysis has been studied,which provides much valuable scientific and applied information for future reference.Chapter one concluded the pathogenic cause of RA and the development of the treatment of RA by oral CⅡ from different sources since the beginning of the last century,which mainly includes the relevant factors in the formation and deterioration of RA,its traditional therapeutic drugs also the OT treatment with CⅡ.It has been proved by quantities of animal experiments that CⅡ from terrestrial animals like chicken,sheep,pig and cow can effectively alleviate the symptoms of RA and shorten its duration.But,considering the epidemic diseases like foot-and-mouth disease(FMD),bovine spongiform encephalopathy(BSE)and Avian Influenza(AI),the aquatic organisms have become the new resources.The shark cartilage contains abundant SCⅡ whose effect on OT treatment of RA remains to be studied.In chapter two,the separation and purification of SCⅡ also the preparation of SCⅡ-P were introduced.SCⅢ was extracted from the cartilage of Prionace glauca by pepsin and then purified.After that,SCⅡ was hydrolyzed using thermolysin.The purity and molecular weight of SCⅡ and SCⅡ-P were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The molecular weight of the α1 chain of SCⅡ was about 130 kDa and about 13 kDa of SCⅡ-P with no other obvious bands,which indicates that the extracted SCⅡ and SCⅡ-p reaching the electrophoretic purity could be used for further experiments.The results by the UV scanning show that SCⅡhas the highest UV absorption at 226 nm,and SCⅡ-P has the highest UV absorption at 232 nm and 277 nm.Chapter three studied several common biochemical properities of SCⅡ and SCⅡ-P.The thermal denaturation temperature of SCⅡ and SCⅡ-P was analyzed by differential scanning calorimetry.The secondary structural characteristics of SCⅡ and SCⅡ-P was analyzed by circular dichroism.The UV-absorption features and IR Spectroscopic Properties of SCⅡ and SCⅡ-P was analyzed by UV scanning and IR spectrometry separately.The antioxidant levels of SCⅡ and SCⅡ-P were analyzed by DPPH method.The results by differential scanning calorimetry shows that the thermal denaturation temperature of SCⅡ is 40.3 ℃,while the SCⅡ-P’s is 19 ℃.Moreover,the salt concentration and hydrogen ion concentration are closely related to their thermal denaturation temperature,of which the impact on SC Ⅱ is much greater than it on SC Ⅱ-P.The results by IR spectrometry show that the main amide of SCⅡ and SCⅡ-P have different absorption band as: 1643 cm-1 and 1630 cm-1 of the amide I,1546 cm-1 and 1552 cm-1 of the amide Ⅱ,1254 cm-1 and 1296 cm-1 of the amide Ⅲ,3403 cm-1 and 3349 cm-1 of the amide A and 2930 cm-1 and 2989 cm-1 of the amide B.Also,both SC Ⅱ and SCⅡ-P have the abilities to scavenge free radicals,which are weaker than that of the existing antioxidants BHT and Vc.Compared to SC Ⅱ,SC Ⅱ-P has better scavenging ability,which may be probably because SC Ⅱ-P has more short peptide residues to provided more electrons that may hinder the radicals of the radicals and convert them into more stable substances by using free radicals.In chapter four,the effects of SCⅡ and SCⅡ-P on the growth of 6T-CEM cells were studied.The results show that both SCⅡ and SCⅡ-P have good biological activity and could induce the death of 6T-CEM.Immunological factors such as FAS / APO-1,viable cells and caspase in the SCⅡ and SCⅡ-P control groups are significantly higher than that in the blank group.The antigen receptor-binding between collagen and 6T-CEM is dose-dependent and the best collagen dose is 10 ug/m L.Collagen contains high level of glycosyl groups which can activate the receptors of FAS,thereby promoting the expression of apoptotic genes in 6T-CEM cell bodies.Fluorescence microscopy proves that SCⅡ-induced apoptosis can lead to cell death in 6T-CEM cells.Meanwhile,the molecular weight and glycation of collagen are also among the key factors of immune tolerance and 6T-CEM cell apoptosis.Chapter five investigated the effects of immune tolerance induced by SCⅡ and SCⅡ-P on the growth of CD4 + T cells and the production of apoptosis factors.The results show that both SCⅡ and SCⅡ-P have effects on the growth of CD4 + T cells.SCⅡ can promote the cell growth under different concentration,while SCⅡ-P is only effective under the specific concentration(>10 ug/ml).It may be due to the differences in the amino acid composition and glycosylation rate of SC Ⅱ and SC Ⅱ-P,which is consistent with the studies before.The immune response of CD4 + T cells are significantly inhibited.The apoptotic factors in the experimental group are much higher than those in the control group,and there is no significant difference between SCⅡ and SCⅡ-P.Thus,we concluded that both SC Ⅱ and SC Ⅱ-P can induce T cell apoptosis,which is similar to the results in previous chapter.According to that,it can be inferred that the establishment,maintenance and regulation of peripheral CD4 + T cell apoptosis and immune tolerance have certain connections,which need to be further verified through the in vivo experiments.In the chapter six,the animal models of rheumatoid arthritis rat induced by Freund’s adjuvant were established,the effect of mouth-induced immune tolerance on RA was evaluated and its mechanism was discussed.SCⅡ and SCⅡ-P were used as the control group with Tripterygium wilfordⅡ polyglycosidium(TWP)as the positive control group,which has been proven to have therapeutic effect on rheumatoid arthritis.SCⅡ and SCⅡ-P proved to be effective to alleviate joint swelling in rats,to weaken the delayed type hypersensitivity(DTH)reaction,to reduce circulating circulatory immune complexes(CIC)in serum and to increase the level of Serum interleukins 10(IL-10).The smoothness of synovial surface of ankle cartilage was observed by high magnification stereomicroscope and tissue section,which shows that both oral SCⅡ and SCⅡ-P can promote the formation of smooth surface of damaged cartilage surface.