ANGIOGENIC FACTORS ASSOCIATED WITH UVEAL MELANOMA

与葡萄膜黑色素瘤相关的血管生成因子

• 批准号: 6518649
• 负责人: ARTHUR S POLANS
• 金额: $ 33.6万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2004-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6518649
• 关键词: RNase protection assay; SDS polyacrylamide gel electrophoresis; angiogenesis; angiogenesis factor; angiostatins; electron microscopy; eye neoplasms; gene expression; human tissue; immunocytochemistry; immunofluorescence technique; in situ hybridization; laboratory mouse; light microscopy; melanoma; metastasis; northern blottings; nucleic acid sequence; polymerase chain reaction; scintillation counter; thromboplastin; tissue /cell culture; uvea; vascular endothelial growth factors; western blottings

Uveal melanoma is the most prevalent primary ocular tumor found in adults. The annual incidence of uveal melanoma is equivalent to the number of new cases of retinitis pigmentosa. The genetic alterations underlying the disease are unknown, and there are few prognostic indicators that can be observed clinically, contributing to enucleation as a principal method of treatment. Unfortunately, events leading to systemic metastases may have occured by the time the ocular symptoms are recognized, and death due to hepatic disease and other complications usually ensues. Clearly, improved methods are needed for the early detection and treatment of the disease. The current application focuses on the contributions of angiogenic growth factors and inhibitors to the progression of the disease. We hypothesize that the malignant and metastatic properties of such tumors depend on the simultaneous expression of multiple angiogenic factors, specifically Cyr61, Tissue Factor, and VEGF. Further, the balance between the expression of these growth factors and inhibitors determines the quiescent period prior to detectable metastases. Specific Aims include: 1. Verify the expression of Cyr61, Tissue Factor and VEGF in primary ocular melanoma using both archival specimens and fresh biopsies of tumor tissue. Methods of detection will include immunohistochemistry, Northern blot hybridization, RT-PCR, and in situ hybridization. Expression will be correlated with vascular density. 2. Assess the importance of Cyr61, Tissue Factor and VEGF during growth of the primary tumor and its metastases using an animal model. Transfected cell lines of uveal melanoma with varying levels of expression of these three genes will be transplanted into the anterior chambers of nude mice, and the growth of the primary ocular tumors, the extent of hepatic lesions, and the vascular density at both sites will be measured. 3. Determine the cellular localization of Cyr61, Tissue Factor and VEGF using a combination of immunocytochemical methods and cellular fractionation. 4. Identify additional angiogenic growth factors (angiopoietin-1) and inhibitors (angiostatin and endostatin) associated with uveal melanoma by RT_PCR, immunoblotting and Northern blot hybridization.

葡萄膜黑色素瘤是成人最常见的原发眼部肿瘤。葡萄膜黑色素瘤的年发病率相当于视网膜色素变性新病例的数量。这种疾病背后的基因改变是未知的，临床上几乎没有可以观察到的预后指标，这有助于摘除眼球作为主要的治疗方法。不幸的是，当眼部症状被发现时，导致全身转移的事件可能已经发生，通常会因肝病和其他并发症而死亡。显然，需要改进方法来及早发现和治疗这种疾病。目前的应用主要集中在血管生成生长因子和抑制物在疾病进展中的作用。我们假设这类肿瘤的恶性和转移特性依赖于多种血管生成因子的同时表达，特别是Cyr61、组织因子和血管内皮生长因子。此外，这些生长因子和抑制因子的表达之间的平衡决定了在可检测到转移之前的静止期。本研究的主要目的包括：1.利用新鲜的眼部黑色素瘤组织标本和档案标本验证Cyr61、组织因子和血管内皮生长因子在眼部原发黑色素瘤组织中的表达。检测方法包括免疫组织化学、Northern印迹杂交、RT-PCR和原位杂交。表达将与血管密度相关。2.利用动物模型评价Cyr61、组织因子和血管内皮生长因子在原发肿瘤及其转移过程中的作用。将这三种基因的不同表达水平的葡萄膜黑色素瘤细胞系移植到裸鼠的前房内，测量原发眼肿瘤的生长情况、肝脏病变的程度以及两个部位的血管密度。3.采用免疫细胞化学和细胞分级相结合的方法确定Cyr61、组织因子和血管内皮细胞生长因子的细胞定位。4.通过RT_PCR、免疫印迹和Northern印迹杂交鉴定与葡萄膜黑色素瘤相关的血管生成因子(Angiopoietin-1)和抑制物(Angiostatin和Endostatin)。