TOMOGRAPHIC/SPECTRAL IMAGING OF FGF-MED ANGIOGENESIS

FGF-MED 血管生成的断层扫描/光谱成像

• 批准号: 2881733
• 负责人: DOROTHEA BECKER
• 金额: $ 18.21万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2881733
• 关键词: angiogenesis; antibody; antisense nucleic acid; apoptosis; athymic mouse; biological signal transduction; cell proliferation; cyanine; fiber optic microscopy; fibroblast growth factor; gene expression; genetic promoter element; growth factor receptors; human tissue; immunocytochemistry; in situ hybridization; melanoma; monophenol monooxygenase; neoplastic cell; nucleic acid repetitive sequence; receptor expression; tissue /cell culture; tomography; vascular endothelium

The overall objective of this proposal is to determine whether tumor angiogenesis constitutes a process that requires uni- or bi-directional signalling between tumor cells and endothelial cells comprising the intratumoral vasculature, and whether this communication is mediated by basic fibroblast growth factor (bFGF) and Fibroblast growth factor receptor (FGFR). To obtain an answer for this important but unanswered question, we will inject human melanomas, grown as subcutaneous tumors in nude mice, with vector constructs generating bFGF/FGFR antisense transcripts under the control of a human tyrosinase promoter and likewise, an RSV LTR promoter. Since tyrosinase is an essential gene of the pigmentation pathway, bFGF and FGFR antisense transcripts, generated under the control of this promoter, will be expressed in melanoma cells but not in tumor-interspersing vascular endothelial cells, In contrast, bFGF/FGFR antisense constructs under the control of the RSV LTR promoter will be expressed in the melanoma cells and the intratumoral vasculature. Thereupon, the tumors will be injected with different cyanine fluorochrome-conjugated antibodies specific for CD31, an antigen expressed on endothelial but not melanoma cells, and S100, an antigen present only melanoma cells. Using fluorescence tomography and AOTF (acousto-optic tunable filters) microscopy, we will then visualize, record and compare, in vivo, the fate of the endothelial and malignant cells within the tumors of the two groups of animals. This schedule of intratumoral inoculations and tumor imaging will be continued until we observe growth-arrest and regression of the bFGF/FGFR antisense-targeted melanomas. After sacrificing the animals and removing their tumors or regressed nodules, we will perform multi-parameter immunohistochemistry and in situ hybridization of tissue sections prepared from the specimens. As the final step of these investigations, the stained melanoma sections will be subjected to qualitative and quantitative microscopic Spectral Imaging to determine the expression or lack thereof, of angiogenesis, proliferation and apoptosis-specific markers.

本提案的总体目标是确定肿瘤血管生成是否构成需要肿瘤细胞和构成肿瘤内脉管系统的内皮细胞之间的单向或双向信号传导的过程，以及这种通信是否由碱性成纤维细胞生长因子（bFGF）和成纤维细胞生长因子受体（FGFR）介导。为了获得这个重要但未回答的问题的答案，我们将在裸鼠中以皮下肿瘤形式生长的人黑素瘤注射在人酪氨酸酶启动子和RSV LTR启动子的控制下产生bFGF/FGFR反义转录物的载体构建体。 由于酪氨酸酶是色素沉着途径的必需基因，因此在该启动子控制下产生的bFGF和FGFR反义转录物将在黑素瘤细胞中表达，但不在肿瘤散布的血管内皮细胞中表达。相反，在RSV LTR启动子控制下的bFGF/FGFR反义构建体将在黑素瘤细胞和肿瘤内脉管系统中表达。因此，将用不同的花青荧光染料缀合的抗体注射肿瘤，所述抗体特异于CD 31（一种在内皮细胞上表达而非黑素瘤细胞上表达的抗原）和S100（一种仅存在于黑素瘤细胞上的抗原）。 使用荧光断层扫描和AOTF（声光可调滤波器）显微镜，我们将可视化，记录和比较，在体内，两组动物的肿瘤内的内皮细胞和恶性细胞的命运。这种肿瘤内接种和肿瘤成像的时间表将继续进行，直到我们观察到bFGF/FGFR反义靶向黑色素瘤的生长停滞和消退。 处死动物并切除肿瘤或消退结节后，我们将对标本制备的组织切片进行多参数免疫组织化学和原位杂交。 作为这些研究的最后一步，将对染色的黑色素瘤切片进行定性和定量显微光谱成像，以确定血管生成、增殖和增殖特异性标志物的表达或缺乏。