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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 conservation of type Ⅱ collagen molecules and the formation of autoantibodies in various animal species. Methods: (1) Type Ⅱ collagen was prepared from different species of animals (including human, swine, cattle and chicken), and the collagen was broken down into multiple fragments with cyanogen bromide. Polyacrylamide gel electrophoresis and Western blotting Westernblot) to identify and measure the characteristics of the antibody. (2) Animal models of rheumatoid arthritis (RA) were established by injection of collagen type Ⅱ into the tail of rats from the subcutaneous to the tail of the rats. Oral type Ⅱ collagen was used to observe the interference with the disease. (3) ELISA analysis of RA patients serum autoantibodies. Results: The positive rates of serum anti-type Ⅱ collagen IgG and IgA antibodies in 112 RA patients were 19.6% and 12.5%, respectively. The positive rates of IgG and IgA in human type Ⅱ collagen were 17.5% and 13.8% respectively. There was no significant difference in the positive rate of IgG and IgA reaction between different species of animals. IgA positive rate higher than the normal rate. Bromide cyanogen can digest all genotypes collagen Ⅱ into 8 ~ 12 fragments, each fragment has a similar molecular weight, with similar site-specific antibodies, CB10, CB11, D and C are the main site of antibodies. The results of animal model showed that the positive rate of RA in pre-oral collagen group was significantly lower than that in model group, with the incidence of 44.4% and 83.3% respectively. The severity of oral collagen group was significantly lower than that of model group and oral collagen group The rate of limb involvement in the model group was 13.8% and 49.9%, respectively. CONCLUSION: The molecular structure of type II collagen of different species is highly conservative. The production of autoantibodies may be caused by the dietary collagen stimulating the immune organs of the human body or directly stimulating the intestinal mucosa. The major locus-specific antibody of patients is CB10 , CB11. In addition, the study also found that D and C are also the major site-specific antibodies, specific site antibodies and the extent of RA disease.