CCR5膜外一、二襻模拟肽的淘选及其对实验性自身免疫性脑脊髓炎小鼠的作用

目的:筛选与CC趋化因子受体5(CC chemokine receptor 5,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。

Screening for simulation peptide specifically binding to the first and the second extra-cellular domain of CCR5 and its therapeutic effect on mice with autoimmune encephalomyelitis

Objective:To screen for simulation peptide binding specifically to the first and the second extra-cellular domain of CC chemokine receptor 5 (CCR5),and to observe their therapeutic effect on mice with experimental autoimmune encephalomyelitis (EAE).Methods: Phage display peptide library was used to screen for peptide sequence binding specifically to CCR5; ELISA was used to identify its binding activity and analyze its DNA sequence. The simulation peptide was synthesized and was injected into abdominal cavity of the EAE mice. Spinal cord tissues were obtained and the pathologic changes were studied by H-E staining in EAE control group and simulation peptide group.Results: Twenty phage clones were randomly chosen for identification and ELISA showed that there were eighteen clones had a strong binding activity with CCR5. The positive clones were sequenced and four peptides of high frequency were obtained: STFTTTL,TPIPQLL,SLPLPKP,and QTSSAAL. Mean clinical score of mice in the EAE model group was 3 and that of the simulation peptide group was 1. H-E staining found that the spinal cord tissues in EAE model group had great number of inflammatory cells and evident demyelination changes; the simulation peptide group had less inflammatory cells and no demyelination changes. The four short peptides had an effect of suppressing and delaying the development of EAE,with the average inhibition rate being 43% (P<0.05).Conclusion: The screened CCR5 simulation peptide has evident inhibitory effect against EAE,indicating that CCR5 may play an important role in the course of multiple sclerosis (MS) .