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: 6441305
• 负责人: DOROTHEA BECKER
• 金额: $ 5.25万
• 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/6441305
• 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.

本研究的总体目标是确定肿瘤血管生成是否需要肿瘤细胞和肿瘤内血管内皮细胞之间的单向或双向信号传递，以及这种信号传递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。为了得到这个重要但尚未解决的问题的答案，我们将在裸鼠皮下生长的黑色素瘤中注射载体构建物，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。由于酪氨酸酶是色素沉积通路的重要基因，因此在该启动子的控制下产生的bFGF和FGFR反义转录本将在黑色素瘤细胞中表达，而不在肿瘤浸润的血管内皮细胞中表达。相反，在RSV LTR启动子的控制下，bFGF/FGFR反义构建物将在黑色素瘤细胞和瘤内血管中表达。然后，将肿瘤注射不同的菁荧光染料偶联抗体，特异性针对CD31和S100。CD31是内皮细胞上表达的抗原，但不存在于黑色素瘤细胞上，S100是仅存在于黑色素瘤细胞上的抗原。利用荧光断层扫描和AOTF（声光可调滤光片）显微镜，我们将观察、记录和比较两组动物肿瘤内内皮细胞和恶性细胞的命运。这种肿瘤内接种和肿瘤成像计划将继续进行，直到我们观察到bFGF/FGFR反义靶向黑色素瘤的生长停止和消退。在牺牲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化和原位杂交。作为这些研究的最后一步，染色的黑色素瘤切片将进行定性和定量显微光谱成像，以确定血管生成、增殖和细胞凋亡特异性标记物的表达或缺乏。