EFFECTS OF NITRIC OXIDE ON FETOPLACENTAL ANGIOGENESIS

描述：(改编自《调查者摘要》)：假设是 碱性成纤维细胞生长因子和血管内皮生长因子诱导的胎儿胎盘血管生成受到部分调控 通过增加NO的产生进而激活MAPK信号 途径，增加碱性成纤维细胞生长因子和血管内皮生长因子的表达，进一步刺激胎儿 胎盘血管生成。三个具体目标将使用 功能良好的OPAE细胞系。目标1：确定外源基因是否 NO刺激的细胞增殖和迁移部分通过 NO供体处理细胞后MAPK信号通路的激活 在没有或存在特定的MAPK激酶抑制剂的情况下，使用 增殖和迁移分析，以及蛋白质分析和蛋白激酶 活性检测MAPK的激活情况。目的II：确定碱性成纤维细胞生长因子和 血管内皮生长因子通过测定总NO增加OFPAE细胞产生NO 使用NO分析仪测定培养基中(亚硝酸盐/硝酸盐)浓度； 如果碱性成纤维细胞生长因子和血管内皮生长因子刺激的细胞增殖和迁移被 部分通过碱性成纤维细胞生长因子和血管内皮生长因子处理细胞激活NO/MAPK通路 在L-NMMA或PD98059/UO126不存在或存在的情况下，使用增殖和 迁移分析、Western分析和激酶活性分析；以及AIM III：至 确定外源性NO或bFGF-和VEGF-增强型NO是否上调bFGF和 三种NO供体和bFGF处理细胞后血管内皮生长因子的表达 和血管内皮生长因子在L-NMMA存在或不存在的情况下使用ELISA法，Western分析和 核糖核酸酶保护试验。

DESCRIPTION: (Adapted from the Investigator's Abstract): The hypothesis is that bFGF- and VEGF-induced angiogenesis in the fetoplacenta is modulated in part via an increase in production of NO which in turn activates the MAPK signal pathway and increases bFGF and VEGF expression, which further stimulates fetal placental angiogenesis. Three Specific Aims will be addressed using the well-characterized OFPAE cell line. AIM 1: to determine if exogenous NO-stimulated cell proliferation and migration are mediated in part via activation of the MAPK signaling pathway by treating cells with the NO donors in the absence or presence of the specific MAPK kinase inhibitor using proliferation and migration assays, as well as Western analysis and kinase activity assays to detect activation of MAPK. AIM II: To determine if bFGF and VEGF increase NO production by OFPAE cells by measuring total NO (nitrite/nitrate) concentrations in the culture media using a NO analyzer; and if bFGF- and VEGF-stimulated cell proliferation and migration are mediated in part via activation of the NO/MAPK pathway by treating cells with bFGF and VEGF in the absence or presence of L-NMMA or PD98059/UO126, using proliferation and migration assays, Western analysis and kinase activity assays; and AIM III: To determine if exogenous NO or bFGF- and VEGF-increased NO upregulates bFGF and VEGF expression by treating cells with three NO donors, as well as with bFGF and VEGF in the absence or presence of L-NMMA using ELISA, Western analysis and RNAse protection assays.