Ⅱ型胶原多肽263～272序列中多个氨基酸替换的意义及其在胶原性关节炎治疗中的作用  

Effect of multiple amino acid substitutions of collagen Ⅱ 263-272 on collagen-induced arthritis

作　　者：李茹 李霞[1] 李萍[1] 姚中强[1] 郭嘉隆[1] 栗占国[1] 

机构地区：[1]北京大学人民医院风湿免疫科,北京100044

出　　处：《北京大学学报（医学版）》2006年第4期360-364,共5页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金资助项目(30271223);北京市自然科学基金资助项目(7041005)~~

摘　　要：目的：研究Ⅱ型胶原变构肽（APL）中氨基酸替换对胶原性关节炎（CIA）的抑制作用，为类风湿关节炎（RA）的多肽免疫治疗提供试验依据。方法：以丙氨酸替换Ⅱ型胶原（CⅡ）263～272多肽中与T细胞受体（TCR）结合的氨基酸，^3H掺人法检测变构肽对RA患者外周血T细胞活化的竞争抑制作用，筛选用于CIA治疗的变构肽。以牛CⅡ诱导大鼠CIA模型，应用不同剂量（1，10，100μg）的CⅡ变构肽每周2次皮下注射，观察大鼠关节肿胀的变化情况。治疗3周后（初次免疫后第35天）处死大鼠，进行X线及病理评分。ELISA法检测血清IFN-γ水平。结果：3条CⅡ263～272变构肽均不同程度抑制CⅡ原型肽对RA患者T细胞的活化。其中，同时替换267位谷氨酰胺（Q）、270位赖氨酸（K）和271位甘氨酸（G）为丙氨酸（A）的APL3效果最好。应用APL3对CIA进行治疗的结果发现，100μg治疗组CIA大鼠的关节炎指数、X线及病理评分均显著低于PBS对照组。血清IFN-γ水平也较PBS对照组明显下降。而无关肽组与PBS对照组相比，关节炎指数、X线和病理评分以及血清IFN-γ水平均无明显差别。结论：TCR结合氨基酸位点替换后的CⅡ263～272多肽具有T细胞低反应性和对原型肽的竞争抑制作用，并可抑制大鼠胶原性关节炎的发展，提示其抑制RA免疫异常的可能性。Objective ： To evaluate the effect of multiple amino acid substitutions of C Ⅱ 263 - 272 peptide on collagen-induced arthritis, and explore a therapeutic strategy of rheumatoid arthritis. Methods. A panel of altered C Ⅱ 263 - 272 peptides was synthesized with substitutions of TCR-contact residues of CⅡ 263 - 272 with alanine. The competitive inhibition of APL to wild type CⅡ 263 - 272 was analyzed by 3 H incorporation assay. CIA model was induced by intradermal injection of bovine CⅡ. Altered CⅡ peptide was injected subcutaneously in different doses （ 1, 10, 100μg, respectively, twice per week） after onset of CIA. The arthritis index, radiologic and histologic scores were recorded to evaluate the severity change of arthritis. Serum levels of IFN-γ were examined by ELISA. Results： Competitive inhibition to wild type CⅡ 263 - 272 of the altered CⅡ 263 - 272 peptides （ APL1, APL2, APL3 ） was found in PBMC from RA patients. Among them, APL3 with substitution of residues 267 （ Q）, 270 （K） and 271 （G） to A showed the most significant effect and thus was used in the treatment of CIA rats. We observed significantly reduced arthritis scores in CIA rats treated with 100μg/dose of APL as compared with rats treated with PBS and peptide control. The mean radiographic and histologic scores was also markedly lower in APL-treated CIA rats than in PBS or peptide control-treated rats. On day 35 after immunization, the serum level of IFN-γ in rats was examined and a significantly low level of serum IFN-γ was found in APL-treated rats. Conclusion： CⅡ 263 -272 peptide with TCR-contact residues substitutions inhibited joint destruction in CIA rats and down-regulated IFN-γ production, suggesting that altered CⅡ peptide might be potentially therapeutic in rheumatoid arthritis.

关 键 词：胶原Ⅱ型 关节炎 类风湿 免疫疗法 

分 类 号：R593.220.4[医药卫生—内科学]