Ⅱ型胶原抗原分子结构的保守性与自身抗体形成关系的研究

Study on relationship between the conservation property of molecular structure of type Ⅱ collagens and their auto-antibody formation

目的:为了解各种属动物Ⅱ型胶原分子结构的保守性和自身抗体形成的关系。方法:(1)从不同种属动物(包括人、猪、牛、鸡)中制备Ⅱ型胶原,用溴化氰把胶原分解成多个片段,以聚丙烯酰胺凝胶电泳和免疫印迹法(Westernblot)鉴定和测定抗体的各项特性。(2)用鸡Ⅱ型胶原自皮下多点注射至大鼠尾部,建立了类风湿关节炎(RA)的动物模型,并通过口服Ⅱ型胶原以观察其对疾病的干扰情况。(3)用ELISA法分析RA病人血清中自身抗体。结果:112例RA病人血清对牛Ⅱ型胶原IgG和IgA抗体反应的阳性率分别为19.6%和12.5%,对人Ⅱ型胶原IgG和IgA反应的阳性率则各自为17.5%和13.8%。对不同种属动物Ⅱ型胶原抗体IgG和IgA反应的阳性率无明显差异。IgA型阳性率高于一般正常比率。溴化氰能把各种属Ⅱ型胶原消化成8～12个片段,每个片段有类似的分子量,有类似的位点特异抗体,CB10、CB11、D和C是主要的位点抗体。动物模型结果表明预先口服胶原大鼠组发生RA的阳性率明显低于模型组,二组的发病率分别是44.4%和83.3%,口服胶原组的病情严重程度明显低于模型组,口服胶原组和模型组的肢体累及率分别为13.8%和49.9%。结论:不同种属Ⅱ型胶原分子结构有很高的保守性,自身抗体的产生可能是饮食胶原刺激人体免疫器官或直接刺激肠道黏膜的免疫系统所致,病人的主要位点特异抗体是CB10、CB11。此外该研究还发现D和C也是主要特异位点抗体,特异位点抗体与RA疾病程度有关。

Objective:To understand the relationship between the conservative property of molecular structure of type Ⅱ collagens of various animal species and their characteristics in auto-antibody formation.Methods:(1)Type Ⅱ collagens prepared from different animals including bovine,porcine,chicken and human were treated with CNBr to segment the collagens into fragments.Polyacrylamide electrophoresis,ELISA and Western blot were used to identify the characteristics of antibodies.(2)Rheumatic arthritis(RA) animal models were produce by injection of type Ⅱ collagen to rat tails subcutaneously.The intervention of disease was observed through oral ingestion of type Ⅱ collagen before injection of collagen.(3)RA patients sera were also analysed with ELISA method.Results:The results of sera analysis of 112 cases of RA patients demonstrated that positive reaction rates to IgG and IgA antibodies of bovine collagen were 19.6% and 12.5% respectively.The positive reaction rates to IgG and IgA antibodies to human collagen were 17.5% and 13.8% respectively.There is no significant difference in positive reaction rates between collagen of different species in the subantibody of IgG and IgA.IgA is higher than normal proportionly.From the results of CNBr treatment,8-12 fragments were obtained from all the type Ⅱ collagens of various species.Corresponding fragments showed similar molecular weights and have similar epitope specific antibody.Among which CB_ 10,CB_ 11,D and C are the prominent epitopes specific antibodies.Results of animal model showed that the incidence rate of RA in rats of the collagen oral administered group is obviously lower than that of the model group.The incidence rates of these two group were 44.4% and 83.3% respectively.The severity of disease of the oral collagen administered group is lighter than the model group and the incidence rates of affected limbs between the oral collagen administered group and the model group were 13.8% and 49.9% respectively.Conclusion:The molecular structure of type Ⅱ collagen of different animal species showed very high conservative property and have considerably similar antigenic properties.The auto-antibody produced in RA patients may be induced by dietary collagen which can stimulate immune organs of human bodies or due to the direct stimulation of immune system in the intestinal mucous menbrane.The man site specific antibodies found in patients were reactive to CB_ 10 and CB_ 11,besides,two main specific epitopes D and C were also found.Epitope specific antibodies are related to severity of RA.