CCR5膜外环特异性结合的活性模拟肽治疗三硝基苯磺酸诱导的SD大鼠结肠炎的分子与细胞免疫机制

In the present study, to use the method of Ph.D-7TM phage display peptide library to screen phage clones of binding specifically the first and the second extracellular loops of CC chemokine receptor 5 (CCR5). Then ten clones of the highest of OD values were tested by ELISA, and four short peptides with seven amino acid of the highest of repeat rate were analyzed and purified. To use the compete and inhibiting experiment of positive phage clones to screen active peptide with compete inhibition and their concentrations in vitro, and use the active short peptides to treat the model of SD rat with colitis induced by TNBS, so as to screen the peptide with therapeutic action in vivo. Then to use the therapeutic peptide to treat the rat with colitis, and to observe effects on symptoms score and pathological score (that was neutrocyte quantitative grading) of inflammatory mucosa of colon, and effects on the levels of TNF-aipha, COX-2 and the number of Th1,Th2,Tc1,Tc2,Treg in their blood and colonic mucosa respectively, so as to investigate the molecular and cellular immune mechanisms of the mimic peptides of CCR5 traeting IBD, and to biuld the basis of the mimic peptides of binding specifically the extracellular loops of CCR5 as a new bioagent treating IBD. Those are remarkable clinical application and marketable value.

本课题拟采用Ph.D-7TM菌体展示肽库技术，以亲和筛选方法筛选与CCR5膜外第1、2襻特异结合的噬菌克隆，通过ELISA鉴定后将OD值最高的10个克隆，经过测序纯化四段重复率极高的七个氨基酸长度的短肽，应用阳性噬菌体克隆的竞争性抑制实验，在体外筛选出有竞争性抑制作用的活性多肽和浓度。构建TNBS诱导的结肠炎SD大鼠模型；并将活性短肽分别应用于模型鼠，从体内筛选出有治疗活性的短肽。利用该活性短肽来干预结肠炎大鼠，观察对大鼠症状评分与炎症肠黏膜的病理评分（即中性粒细胞定量分级）的影响，对大鼠血液与肠道黏膜中的TNF-alpha、COX-2和Th1、Th2、Tc1、Tc2、Treg细胞数的影响，探讨CCR5模拟肽对IBD治疗的分子细胞免疫机制。为CCR5膜外环特异性结合的模拟肽作为一种新型的生物制剂治疗IBD奠定基础，具有临床应用价值与市场前景。

开发针对新靶点CCR5的生物治疗是IBD治疗研究的热点问题。我们在国家自然科学基金（81370499）资助下，已完成了CCR与IBD的研究概况的系统综述；在体外观察不同浓度的生物制剂Infliximab对SD大鼠骨髓来源的mesenchymal stem cells（MSC）增殖、凋亡和细胞周期无显著性的影响；CCR5在IBD活动期组肠黏膜呈高表达，IBD活动期肠黏膜的中性粒细胞、嗜酸性粒细胞及淋巴细胞明显增多，淋巴细胞分级与CCR5表达呈正相关性，β-arrestin2在IBD活动期肠黏膜呈明显低表达，与CCR5表达呈负相关性；利用噬菌体肽库展示技术淘选与大鼠CCR5膜外第一、二胞外环特异性结合的短肽ECL1与ECL2，在体外初步鉴定其与CCR5的结合能力，并在TNBS诱导的SD大鼠结肠炎模型上初步证实有治疗作用。正在进行的研究内容：两条短肽ECL1与ECL2注射后对SD大鼠炎症肠黏膜的病理评分的影响（标本已收集，但未阅片评分）；观察SD大鼠血液和大肠黏膜的TNF-alpha、COX-2、Th1、Th2、Tc1、Tc2、Treg的检测（标本已收集，检测实验仍在进行中）。本课题已取得了初步的结果，能按计划按进度完成基本的研究内容。说明了CCR5可作为IBD治疗的新靶点，为CCR5膜外环特异性结合的模拟肽作为一种新型的生物制剂治疗IBD奠定基础，具有临床应用价值与市场前景。

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