TUMOR-SECRETED VASCULAR PERMEABILITY FACTOR

肿瘤分泌的血管通透性因子

• 批准号: 3186473
• 负责人: DONALD R SENGER
• 金额: $ 20.4万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-01-01 至 1994-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3186473
• 关键词: antibody formation; antibody neutralization test; athymic mouse; body fluids; calcium; gene expression; guinea pigs; high performance liquid chromatography; human tissue; immunologic techniques; in situ hybridization; inflammation; injection /infusion; laboratory rabbit; malignant ascites; messenger RNA; molecular cloning; monoclonal antibody; neoplastic cell; neoplastic growth; neoplastic transformation; northern blottings; pleural neoplasms; protein purification; protein sequence; protein structure function; synthetic peptide; tissue /cell culture; transposon /insertion element; vascular endothelium; vascular endothelium permeability

The long-term objectives of this project are to elucidate the biological functions of vascular permeability factor (VPF) and, in particular, to define the significance of VPF for tumor biology. The Specific Aims of this proposal are 1) to further define VPF structure, including complete amino acid sequence (as deduced from cDNA sequence), and to derive antibodies to synthetic peptides which block VPF activity; 2) to study expression of VPF and VPF mRNA by a wide variety of tumor and normal cells in vivo and in vitro; and 3) to study the long-term effects of VPF on tumor growth, fluid accumulation and endothelium in vivo. cDNA inserts encoding human and guinea pig VPF from lambda-gt11 clones (identified with antibodies to VPF N-terminal peptide) will be subcloned and sequenced, and from these nucleotide sequences, complete VPF amino acid sequence will be derived. Specific peptides (approximately 25 amino acids), representing limited portions of the entire VPF sequence, will be used as immunogens to obtain antibodies which recognize epitopes throughout the VPF molecule. Anti-peptide antibodies which neutralize VPF activity will allow for identification of important functional sites within the molecule. Expression of VPF and VPF mRNA by a wide variety of tumor and normal cells in vivo and in vitro will be studied with highly sensitive methods. Expression of VPF mRNA in vivo will be detected with in situ hybridization. Expression of VPF activity by cells in vitro will be detected with an endothelial cell/cytosolic calcium assay, which is at least 300-times more sensitive than both the Miles dermal vessel permeability assay and our currently available conventional immunoassays for VPF. Comparisons (in vitro) between expression of VPF activity and cellular VPF mRNA levels will be made in conjunction with Northern blotting. Finally, the biological effects of VPF in vivo will be characterized with particular emphasis on determining the significance of VPF expression for tumor growth and fluid accumulation in the peritoneal cavity. To determine the effects of VPF on tumor growth and fluid accumulation, animals bearing ascites tumors will receive repeated intraperitoneal injections of VPF-neutralizing antibodies. To determine the effects of purified VPF (in the absence of tumor) on the vessels and other tissues of the peritoneal wall, animals will receive frequently-repeated injections of purified VPF. The goal will be to determine if any of the profound changes that commonly occur in the peritoneal wall of ascites tumor-bearing animals (e.g., edema, fibrin deposition, angiogenesis, and fibrosis) occur in response to frequently repeated administration of purified VPF.

该项目的长期目标是阐明生物学 血管通透性因子（VPF）的功能，特别是 明确VPF在肿瘤生物学中的意义。具体目标是 建议1）进一步定义VPF结构，包括完整的氨基 酸序列（从cDNA序列推导出），并获得针对 阻断VPF活性的合成肽; 2）研究VPF的表达 和VPF mRNA的多种肿瘤和正常细胞在体内和在 体外; 3）研究VPF对肿瘤生长、液体 体内蓄积和内皮。 来自Aphroda-gt 11克隆的编码人和豚鼠VPF的cDNA插入片段 （用VPF N-末端肽的抗体鉴定）将被亚克隆 并进行测序，并从这些核苷酸序列中获得完整的VPF氨基酸 序列将被导出。特异性肽（约25个氨基酸）， 表示整个VPF序列的有限部分，将被用作 免疫原以获得识别整个VPF中的表位的抗体 分子。中和VPF活性的抗肽抗体将允许 用于鉴定分子内的重要功能位点。 多种肿瘤和正常细胞表达VPF和VPF mRNA 将采用高灵敏度的方法进行体内和体外研究。 用原位杂交法检测VPF mRNA在体内的表达。 体外细胞的VPF活性表达将用免疫组织化学方法检测。 内皮细胞/胞质钙测定，其至少是300倍以上 比Miles皮肤血管渗透性测定和我们的 目前可用于VPF的常规免疫测定。比较（以 VPF活性表达和细胞VPF mRNA水平之间的关系将 结合北方印迹法进行。 最后，VPF在体内的生物学效应将被表征， 特别强调确定VPF表达的意义， 肿瘤生长和腹腔积液。以确定 VPF对肿瘤生长和体液积聚的影响，荷瘤动物 腹水肿瘤将接受重复的腹膜内注射 VPF中和抗体。为了确定纯化的VPF（在 没有肿瘤）在腹膜的血管和其它组织上 动物将接受纯化VPF的频繁重复注射。 我们的目标将是确定是否有任何深刻的变化， 发生在腹水肿瘤携带动物的腹膜壁（例如，水肿， 纤维蛋白沉积、血管生成和纤维化）响应于 经常重复给予纯化的VPF。