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目的:筛选与CC趋化因子受体5(CC chemokine receptor5,CCR5)第一、二膜外襻特异结合的模拟肽,并将其应用在实验性自身免疫性脑脊髓炎的小鼠模型(experimental autoimmune encephalomyelitis,EAE),来观察使用模拟肽后EAE模型小鼠的治疗效果。方法:用噬菌体随机7肽库筛选与CCR5特异结合的多肽序列,用ELISA鉴定其结合活性并进行DNA测序分析;接着合成与其氨基酸序列一致的模拟肽,腹腔内注射至EAE小鼠。取小鼠脊髓组织,通过H-E染色,观察模拟肽组和EAE对照组小鼠脊髓组织病理学改变。结果:随机挑出20个噬菌体克隆进行鉴定,ELISA分析显示其中18个克隆与CCR5有较强的结合活性,将其阳性噬菌体克隆进行测序,从中得到重复率极高的4段小分子肽,它们分别为STFTTTL(SL)、TPITQLL(TL)、SLPLPKP(SP)、QTSSAAL(QL)。对照组EAE小鼠的平均临床评分为3分,模拟肽组EAE小鼠的平均临床评分为1分。H-E染色后,经病理分析EAE组小鼠的脊髓内有大量的炎症细胞浸润,白质脱髓鞘改变明显。而模拟肽组小鼠有较少的炎症细胞浸润,且没有白质脱髓鞘改变。4段短肽分别具有抑制、延迟发生EAE的作用,其平均抑制率为43%(EAE组为83%,两者比较有统计学差异,P<0.05)。结论:筛选的CCR5模拟肽具有对EAE明显的抑制作用,提示CCR5可能在EAE的发病进程中发挥着重要的作用。
OBJECTIVE: To screen the peptidomimetic peptides that bind specifically to the first and second extrahepatic plaques of CC chemokine receptor 5 (CCR5), and to apply them in a mouse model of experimental autoimmune encephalomyelitis autoimmune encephalomyelitis, EAE) to observe the therapeutic effect of EAE model mice using the mimetic peptide. Methods: Peptide sequence specific binding to CCR5 was screened by phage random 7-peptide library. The binding activity of CCR5 was identified by ELISA and DNA sequencing analysis was performed. Then the mimetic peptide with the same amino acid sequence was synthesized and injected intraperitoneally into EAE mice. The spinal cord tissues of mice were taken out and stained with H-E to observe the histopathological changes of the spinal cord in mimetic peptide group and EAE control group. RESULTS: Totally 20 phage clones were randomly selected and identified. ELISA analysis showed that 18 of the clones had strong binding activity with CCR5. The positive phage clones were sequenced to obtain 4 small peptides with high repetition rate. They are STFTTTL (SL), TPITQLL (TL), SLPLPKP (SP) and QTSSAAL (QL) respectively. The mean clinical score of the EAE mice in the control group was 3 points and that of the EAE mice in the mimetic peptide group was 1 point. H-E staining, pathological analysis EAE mice spinal cord in a large number of inflammatory cell infiltration, white matter demyelination changed significantly. Mock peptide mice had less infiltration of inflammatory cells and no white matter demyelination. The 4-step short peptide inhibited and delayed EAE, with an average inhibition rate of 43% (83% in EAE group, statistically significant difference between the two groups, P <0.05). Conclusion: The selected CCR5 mimic peptide has a significant inhibitory effect on EAE, suggesting that CCR5 may play an important role in the pathogenesis of EAE.