ANTISENSE OLIGONUCLEOTIDES AS INHIBITORS OF TUMOR-INDUCED ANGIOGENESIS

反义寡核苷酸作为肿瘤诱导的血管生成的抑制剂

• 批准号: 5201581
• 负责人: J A LAUTENBERGER
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5201581
• 关键词: angiogenesis; antisense nucleic acid; cell line; cell migration; cell motility; growth inhibitors; hybrid cells; messenger RNA; neoplastic process; oligonucleotides; oncoproteins; phenotype; protein structure function; protooncogene; simian virus 40; tissue /cell culture; transcription factor; transfection; vascular endothelium

It was determined that the endothelial cell-specific cytokine vascular endothelial growth factor (VEGF) increased the level of ETS1 mRNA by two- to four fold. We have also determined that ETS1 protein level in VEGF-treated endothelial cells was increased. These observations have been made in both human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells (HMVEC-L). The response of these cells to VEGF occurs within 4 hours of treatment and occurs at an optimal concentration of 10-20 ng/ml VEGF. In order to determine a direct role for ETS1 in angiogenesis-associated endothelial cell phenotype, cells were treated with antisense oligonucleotides using lipofectin as a carrier. After treatment, the cells were assayed for their ability to migrate through matrigel-coated membranes. We have found that antisense ETS1 oligonucleotides inhibits this migration up to 75%. Sense or random sequence oligonucleotides had no effect. There was only a slight reduction in the level of ETS1 mRNA in the antisense-treated cells. However, antisense oligonucleotides lowered the level of u-Pa mRNA. For immortalization, HUVEC and HMVEC-L have been infected by Adeno12-SV40 hybrid virus (Ad12-SV40), amphotropic retroviruses expressing HPV-16 E6 and E7, or transfected with the HPV-18 genome. They expressed Factor VIII-related antigen and integrin alpha/v/beta3, take up fluorescent-acetylated low density lipoprotein (Dil-Ac-LDL), and develop capillary-like tube structures upon reaching confluence, indicating that the immortalized cells retain endothelial cell-specific features.

已经确定，内皮细胞特异性细胞因子血管内皮细胞 内皮生长因子（VEGF）使ETS 1 mRNA水平增加了2倍， 四倍。 我们还确定了ETS 1蛋白水平， VEGF处理的内皮细胞增加。 这些观测 在人脐静脉内皮细胞（HUVEC）和 人肺微血管内皮细胞（HMVEC-L）。 的响应 这些细胞向VEGF的转化发生在治疗后4小时内， VEGF的最适浓度为10-20 ng/ml。 为了确定ETS 1在血管生成相关的血管生成中的直接作用， 内皮细胞表型，细胞用反义核酸处理 使用脂质体作为载体的寡核苷酸。 治疗后 测定细胞迁移通过基质胶包被的 膜。 我们已经发现反义ETS 1寡核苷酸抑制 迁移率高达75%。 有义或随机序列寡核苷酸具有 没有效果。 ETS 1 mRNA水平仅略有下降， 在反义处理过的细胞中。 然而，反义寡核苷酸 降低u-Pa mRNA水平。 为了永生化，HUVEC和HMVEC-L已经被Adeno 12-SV 40感染 杂合病毒（Ad 12-SV 40），表达HPV-16 E6的嗜异性逆转录病毒 和E7，或用HPV-18基因组转染。 他们表示， VIII相关抗原和整合素α/v/β 3，摄取 荧光乙酰化低密度脂蛋白（Dil-Ac-LDL），并开发 达到汇合时的毛细管状管结构，表明， 永生化细胞保留内皮细胞特异性特征。