出国医疗

人体结肠癌血管绑定多肽的筛选和鉴定

来源:万方数据    时间:2014-02-10 15:30  浏览次数:

  Selection and identification of a specific binding peptide targeting to blood vessel of human colon cancer

  目的 建立小鼠结肠癌模型,筛选并鉴定能够与人结肠癌血管内皮细胞特异结合的噬菌体呈现短肽.

        方法 建立小鼠荷瘤模型.鉴定阳性噬菌体的体内归巢能力及内皮细胞结合能力.人工合成噬菌体呈现短肽,通过竞争结合实验观察短肽对其呈现噬菌体的竞争结合抑制作用.并检测短肽与共培养内皮细胞及结肠癌血管的特异性结合能力。

        结果 鉴定出两种外源肽噬菌体,称为pCV1及pCV2.体内实验显示,pCV1、pCV2均能特异性归巢于人结肠癌移植瘤组织,滴度比值 pCV1/pCont、pCV2/pCont分别为15.9、20.1倍,明显高于对照组织.体外细胞酶联免疫吸附试验(ELISA)结果显示pCV2在 Co-HUVECs上的结合显著高于在HUVECs上的结合,其比值达2.61.人工合成短肽CV2能特异性竞争抑制pCV2向结肠癌移植瘤内的归巢及与 Co-HUVECs的特异性结合.免疫荧光染色结果显示,FITC-CV2特异性结合于Co-HUVECs的胞膜与核周胞质,以及结肠癌移植瘤的血管组 织。

        结论 得到两个能特异性结合于结肠癌移植瘤的噬菌体单克隆pCV1及pCV2.pCV2能够特异性结合于Co-HUVECs,pCV2所呈现的环状九肽CV2介 导了它与Co-HUVECs的特异结合.短肽CV2具有与Co-HUVECs及结肠癌移植瘤血管特异性结合的能力,有可能用于结肠癌的血管靶向治疗.

  Abstract:

  Objective To select, identify and analyze a peptide binding specifically to blood vessels of human colon cancer by phage displayed peptide library in vivo. Methods Animal models were established using sub-renal capsular assay (SRCA) in immunosuppressive mice implanted with human colon cancer xenografts. The phage displayed peptide library was injected intravenously into mice. After 4 rounds of selection, 20 clones were picked up randomly and sequenced individually. The homing ability to human colon cancer xenografts and co-cultured human umbilical vein endothelial cells ( Co-HUVECs with human colon cancer Lovo cells) of the positive phage clones were determined by in vivo binding assay and in vitro cell enzyme linked immunosorbent assay ( ELISA ). The binding ability to Co-HUVECs of peptide-displayed phage clone was identified by immunocytochemical stain. Peptide displayed on the phage was synthesized and competitive binding assays were performed to observe the competitive inhibition effect of the peptide with their phage counterpart.

  Immunofluorescence microscopy was used to study the binding of synthesized peptides to Co-HUVECs and vascular vessels in colon cancer.

  Results Two peptide sequences were obtained finally and named CV1 and CV2. In vivo binding assay showed that the homing ability to human colon cancer xenografts of peptides of CV1 or CV2 was higher than that of control organs. In vitro cell ELISA suggested that CV2 phage preferably binded to Co-HUVECs rather than control HUVECs. Then CV2 phage clone was identified further. Immunocytochemical staining revealed that CV2 phage preferably binded to Co-HUVECs rather than the control. Competition binding assays demonstrated a significant competition between the synthesized peptide CV2 and the phage displaying CV2 while binding to Co-HUVECs or human colon cancer xenografts. Under the immunofluorescence microscopy, fluorescence-labeled CV2 peptide was seen on the membrane and in the perinuclear cytoplasm of Co-HUVECs, and bound to colon cancer xenografts rather than control organs. Conclusion Two phage clones displaying CV1 and CV2 peptide could target to human colon cancer xenografts. The peptide CV2 and its displayed phage were identified binding preferably with Co-HUVECs. And the cyclic nonapeptide (CV2) was binding site of the CV2 phage with Co-HUVECs. Synthesized nonapeptide CV2 had specificity to Co-HUVECs and colon cancer vascular endothelial cells. The peptide CV2 could be used in target therapy of tumor angiogenesis.

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