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目的 研究人类白细胞抗原 (HLA) DRβ1特异性Ⅱ型胶原 (CII)多肽的T细胞受体(TCR)结合表位改变对T细胞激活的影响。探讨通过抑制T细胞对抗原肽的识别治疗类风湿关节炎的新途径。方法 利用AutoDock3 0系统设计非T细胞结合肽 ;以激光共焦显微镜及流式细胞仪观察荧光标记的CⅡ 2 6 3 2 72修饰肽的细胞内转运及其在细胞膜上的表达 ;应用HLA DR1转基因抗原呈递细胞和特异性T细胞的激活体系 ,研究替换TCR识别氨基酸的CII修饰肽在T细胞激活中的作用。结果 计算机模拟显示CII第 2 6 3位苯丙氨酸 (F)、2 6 6位谷氨酸 (E)是与HLA DR1结合的关键位点 ,而 2 6 7位谷氨酰胺 (Q)、2 6 9位脯氨酸 (P)和 2 70位赖氨酸 (K)是T细胞识别的主要功能氨基酸。用甘氨酸 (G)、和 /或丙氨酸 (A)替换上述位点可去除功能性侧链。CⅡ修饰肽可被HLA DR1转基因抗原呈递细胞摄取并与膜表面的HLA DR1分子结合。CⅡ 2 6 3 2 72原型肽可激活T细胞 ,而用A或G单一替换 2 6 7、2 6 8、2 6 9、2 70位氨基酸或连续替换 2 6 8 2 70位氨基酸的修饰肽对T细胞的激活能力明显降低 (P <0 0 1)。低反应性修饰肽 2 70A、sub2 6 8 2 70对CⅡ诱导的T细胞激活有显著抑制作用。结论 通过替换CII中TCR特异性氨基酸可减弱或消除其对T细胞的结合能力及激?
Objective To investigate the effect of T cell receptor (TCR) binding epitope of DRβ1-specific type Ⅱ collagen (CII) polypeptide on the activation of T lymphocytes in human leukocyte antigen (HLA). To explore a new way to treat rheumatoid arthritis by inhibiting the recognition of antigen peptides by T cells. Methods The non-T cell binding peptide was designed by using AutoDock30 system. The intracellular transport of fluorescently labeled CⅡ 2 3 2 72 peptide and its expression on the cell membrane were observed by laser scanning confocal microscope and flow cytometry. The HLA DR1 transgene Antigen presenting cells and specific T cell activation system to study the role of CII modified peptides replacing TCR recognition amino acids in T cell activation. Results Computer simulations showed that the phenylalanine (F) at position 263 of CII and glutamate (E) at position 266 were the key sites for binding to HLA DR1, while the glutamine (Q) at position 267, The 269th proline (P) and 270th lysine (K) are the major functional amino acids recognized by T cells. Functional sites can be removed by replacing the sites with glycine (G), and / or alanine (A). C II -modified peptides can be taken up by HLA DR1 transgenic antigen-presenting cells and bound to membrane-surface HLA DR1 molecules. CII 2 6 3 2 72 The prototype peptide activates T cells while a single substitution of A or G with a modified peptide pair of 267, 268, 269, 270 or a continuous substitution of amino acids 268270 T cell activation was significantly reduced (P <0.01). Low-reactivity modified peptide 2 70A, sub 2 6 8 2 70 significantly inhibited CⅡ-induced T cell activation. Conclusion By substituting the TCR-specific amino acids in CII, the ability of binding to T cells can be reduced or eliminated.