摘要
背景:肿瘤细胞高表达分子被作为肿瘤靶向治疗的药物靶点。肿瘤靶向治疗有三种研究策略。第一种策略是获得肿瘤药物靶点的单克隆抗体。第二种策略是噬菌体肽库筛选获得与药物靶点分子高亲和力结合的小分子肽。第三种策略是获得生长因子片段及其衍生物,以阻断生长因子受体的功能。
胰岛素受体是一种生长因子受体,在肝癌细胞表面表达高于正常肝细胞,是肝癌靶向治疗的侯选靶点分子之一。噬菌体肽库是在一种噬菌体外壳蛋白末端连接上大量结构各异的肽链。当肽库与胰岛素受体结合,只有与受体具有高亲和力的多肽被筛选出来。通过提取噬菌体DNA测序获得多肽的氨基酸残基序列。再用多肽合成仪人工合成多肽。这种多肽可能具有肝癌等肿瘤靶向性。用噬菌体肽库筛选肿瘤高表达分子的高亲和力多肽是研究肿瘤靶向药物的一条重要途径。
目的:噬菌体随机环7肽库筛选能与胰岛素受体高亲和力结合的多肽,人工合成多肽作为放射性核素或肿瘤化疗药物载体,用于肝癌的放射性核素显像诊断和肝癌的靶向治疗。
方法:筛选:将大鼠肝胰岛素受体包被在培养皿上,用噬菌体环7肽库与胰岛素受体结合,收集结合在固相受体上的噬菌体。扩增筛选获得的噬菌体后用于下一轮筛选。通过数轮筛选,获得与受体亲和力高的噬菌
BACKGROUND: Overexpression of growth factor receptors and some glycoprotein has been implicated in most of the tumors. The over expressed molecules of the tumor cell are the best sites in tumor targeting and anticancer therapies. Three methods could be employed in tumor targeting therapy. The first is to generate a monoclonal antibody against tumor high expressed molecules. The second is to select a high affinity peptide of the growth factor receptors or glycoprotein from the phage display peptide library. The third is to obtain an analogue or antilogous of the growth factors. Insulin receptors, a kind of growth factor receptors over expressed on the surface of the hepatocellular carcinoma (HCC) cells, have become potential targeted sites in HCC therapy. Phage display peptide library is a vast library of random peptide sequences expressed as fusions with coat proteins of bacteriophages. An in vitro selection process could elute the specifically-bound phages of insulin receptors. Individual phage clones are characterized by DNA sequencing. A new- peptide is synthesized
corresponding to the DNA sequence. The new peptide will be a potential HCC-targeting molecular.AIM: The purpose of this study is to look for peptide ligands of insulin receptors by a bio-panning of a disulfide constrained phage display peptide library. We hypothesized that the peptides will play an important role in HCC diagnostic imaging and/or targeting therapy.METHODS: Rat liver insulin receptors were immobilized on a polystyrene plate by incubating in 0.1mol/L NaHCO3 overnight at 4℃. Panning was carried out by incubating a library of phage- displayed peptides with immobilized insulin receptors for 40min at room temperature. The bound phages were eluted with 0.2M glycine-HCl and amplified with 200μl Ecol.ER2738 in 20ml LB-Medium in a 250ml Erlenmeyer flask. After three round of bio panning, 12 microphage colons were picked out to amplify. 11 clones were finally sequenced according to the ELISA.A new disulfide constrained peptide CY-10 (CQSKHWRHCY) was synthesized corresponding to insert sequence. The apparent affinity constant of 125I-CY-10 binding to insulin receptors was revealed by saturation binding assay and Scachard analysis.The radioactivity of 125I-CY-10 bound to SMMC 7721 cells to the L-02 cells was compared first. Then the bio distribution of 125I-CY-10 in mice bearing human tumor was conducted following the administration of the 125I-CY-10 at 5,15,60,180,360,900min. The whole body posterior image of 131I-CY-10 in mouse bearing tumor performed 24 hr post injection. RESULTS: After three rounds, enzyme-linked immunosorbent assay (ELISA) was employed to investigate 12 individual clones bound with the insulin
receptors. All of the 12 clones were ELIS A positive. We picked out 11 clones that with a high OD value for DNA sequence. The results shows that all of the 11 colons have the same insert oligonucletide sequences ATGACGCCAATGCTTCGACTG. According to the oligonucletide sequence, a new peptide CQSKHWRHC-Y (named CY-10) was synthesized. The apparent affinity constant of 125I-CY-10 (Kd value) bind with insulin receptor is 8.909 ×10-8mol/L. The insulin receptor exhibits a 1.715-fold affinity for CY-10 than for insulin.The HCC SMMC 7721 cells bound much more 125I-CY-10 than L-02 cells. The biodistribution shows that the hepatocellular carcinoma tissue of the mice bearing human SMMC 7721 tumors has a higher uptake of 125I-CY-10 than the muscle, the bone and the brain. The 125I-CY-10 activities in tumor tissue reach maximum peak values at 3hr post-injection. The ratio of tumor to muscle at 3 hour is 2.2218. The whole body imaging of 131I-CY-10 shows a high tumor uptake.CONCLUSSION: This study generated a high-affinity peptide of insulin receptor. The uptake of peptide CY-10 in HCC tumor tissue of the naked mice uptake is high. CY-10 could be a potent targeted reagent of HCC.
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