EFFECTS OF NITRIC OXIDE ON FETOPLACENTAL ANGIOGENESIS

描述：（改编自研究者摘要）：假设是， bFGF和VEGF诱导的胎儿胎盘血管生成部分受到调节 通过增加NO的产生，这反过来又激活MAPK信号 增加bFGF和VEGF的表达，这进一步刺激了胎儿的生长。 胎盘血管生成三个具体目标将通过使用 良好表征的OFPAE细胞系。目的1：确定外源性 一氧化氮刺激的细胞增殖和迁移部分是通过 通过用NO供体处理细胞激活MAPK信号通路 在不存在或存在特异性MAPK激酶抑制剂的情况下， 增殖和迁移测定，以及Western分析和激酶 活性测定以检测MAPK的活化。目的II：确定bFGF和 通过测定总NO，VEGF增加OFPAE细胞的NO产生 使用NO分析仪测定培养基中的亚硝酸盐/硝酸盐浓度;以及 如果bFGF-和VEGF-刺激的细胞增殖和迁移被介导， 部分通过用bFGF和VEGF处理细胞来激活NO/MAPK通路 在不存在或存在L-NMMA或PD 98059/UO 126的情况下，使用增殖和 迁移测定、蛋白质印迹分析和激酶活性测定;以及AIM III： 确定外源性NO或bFGF和VEGF增加的NO是否上调bFGF， 用三种NO供体以及bFGF处理细胞的VEGF表达 和VEGF在不存在或存在L-NMMA的情况下使用ELISA，Western分析和 RNA酶保护测定。

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.