TCP-1靶向多肽在胃癌肿瘤血管上的特异靶点鉴定及其靶向传输抗肿瘤药物的研究

The aims of this study are to identify the binding molecule in gastric tumor blood vessels and develop a biomarker and a drug target to enhance chemotherapy. Using phage display biopanning technology, our previous study identified a small peptide (CTPSPFSHC) termed TCP-1 that specifically bound to blood vessels of tumors induced orthotopically by a human gastric cancer cell line (MKN45) but not to other tissues or subcutaneous MKN45 xenograft in mice. These findings suggest that there is a specific binding site in tumor blood vessels. With these findings, identification of this molecule could have significant clinical implications in the treatment of gastric cancer (GC). In this regard, the binding molecule could be a specific biomarker for GC and used as a molecule for drug targeting during chemotherapy. Our preliminary data also showed that the same binding capacity of TCP-1 was found on the cell membrane of MKN45. With this important information, we shall use TCP-1 to pull down the binding molecule from these membranes, in order to identify and also define the properties of this molecule. The identity of this binding molecule will be confirmed in the gastric tumor vasculature in animals. This molecule will be a guide for GC treatment using TCP-1 as a carrier for anticancer drugs in targeted drug therapy. In this study, we shall employ tumor necrosis factor alpha (TNF-alpha) which has been identified to have potent anti-angiogenic activity but possess severe systemic toxicity. We shall couple TCP-1 with TNF-alpha using recombinant technology in the hope that new anticancer agents for GC could be developed. This TCP-1/TNF-alpha could also enhance the efficacy of other clinical anticancer drugs, e.g. 5-fluorouracil and irinotecan through destruction and removal the vascular barrier and/or stabilization of the tumor vascular system. These novel therapeutic strategies will not only enhance the anticancer action of TNF-alpha on tumor blood vessels but also produce synergistic effects with other anticancer drugs together with lower systemic toxicity. This could greatly increase the viability of chemotherapy in humans. Identification of the binding molecule for TCP-1 at tumors is useful for targeted and guided therapy for GC. This serves as a good example for personalized medicine in cancer patients.

本研究旨在研究靶向TCP-1多肽在胃癌肿瘤上的结合位点，以及将其开发应用为肿瘤的生物指标和增强化疗药物的靶标。利用噬菌体展示实验，我们分离鉴定了一个小分子多肽并命名为TCP-1。该多肽特异性地靶向于由人胃癌细胞株MKN45诱导的原位胃癌组织的血管上，而非其他正常组织器官。这些结果表明肿瘤血管上存在某个特异的靶点。该靶点可被用作胃癌早期检测的生物指标及化疗药物靶向传输的靶点。我们将运用蛋白质交互作用沉淀试验（Pull-down assay）寻找到TCP-1在MKN45细胞膜上的特异靶点，并深入研究该靶点。该靶点的鉴定将会给胃癌的治疗带来新的方向──利用TCP-1多肽作为药物载体靶向给药。本项目中，我们将运用基因重组技术耦合TCP-1多肽和TNF-alpha以期开发出新的靶向制剂用于治疗胃癌等癌症。TCP-1多肽在肿瘤组织上的结合位点的鉴定将有助于靶向治疗胃癌，也会给癌症病人的个体化用药。

本项目采用MKN45 细胞及小鼠原位胃癌模型为对象，研究TCP-1 多肽与肿瘤细胞特异结合的机制以及在肿瘤的靶向治疗中的应用。重点研究TCP-1 多肽在肿瘤细胞和肿瘤血管上的结合位点以及运用TCP-1 多肽/TNF-alpha 复合物治疗原位胃癌或/和增强化疗药物的疗效。主要内容包括：.（一）TCP-1 多肽在胃癌组织上靶向结合位点的研究。本项目按照研究计划，研究靶向TCP-1 多肽在胃癌肿瘤上的结合位点，以及将其开发应用为肿瘤的生物指标和增强化疗药物的靶标。利用噬菌体展示实验，我们分离鉴定了一个小分子多肽并命名为TCP-1。该多肽特异性地靶向于由人胃癌细胞株MKN45 诱导的原位胃癌组织的血管上，而非其他正常组织器官。这些结果表明肿瘤血管上存在某个特异的靶点。该靶点可被用作胃癌早期检测的生物指标及化疗药物靶向传输的靶点。我们运用蛋白质交互作用沉淀试验（Pull-down assay）寻找到TCP-1在MKN45 细胞膜上的特异靶点，并深入研究该靶点。研究结果显示，TCP-1多肽而非对照多肽可特异性靶向一分子量约为36 kDa 的蛋白分子，该分子经高分辨质谱鉴定结合蛋白数据库分析得出其很可能为一个参与钙离子调节的分子A。该靶点的鉴定将会给胃癌的治疗带来新的方向──利用TCP-1多肽作为药物载体靶向给药。.（二）靶向TCP-1 多肽在胃癌治疗中的应用。在我们前期的研究中，已经证明了TCP-1在小鼠体内注射的安全性。本项目中，我们还运用基因重组技术耦合TCP-1 多肽和TNFα以期开发出新的靶向制剂用于治疗胃癌。以MKN45 细胞为对象，皮下接种得到胃癌组织，手术植入小鼠胃部，构建小鼠原位胃癌模型。利用DNA 重组技术构建TCP-1 多肽和TNFα复合物，在小鼠原位胃癌模型中研究TCP-1 多肽偶联TNFα复合物的抗血管生成及其抗肿瘤活性。构建TCP-1 多肽偶联TNFα复合物联合临床胃癌用药，如5-氟脲嘧啶和伊立替康在小鼠原位胃癌模型中研究了该联合用药方案的有效性。