人sflt-1因片段转染对缺氧、高糖下内皮细胞vegf的erk12、pi3kakt信号通路的影响.pdfVIP

人sFlt-1 基因片段转染对缺氧、高糖下内皮细胞 VEGF 的ERK1/2、PI3K/Akt 信号通路的影响 摘 要 目的 比较sFlt-1受体胞外第2－3区和第2－4区对缺氧、高糖条件 下血管内皮细胞增殖、迁移、视网膜血管通透性改变及p44/p42MAPK、 PI3K/Akt信号通路的影响。 方法 以RT-PCR法从人胎盘组织cDNA文库中扩增出sFlt-1胞外第2 －3区和第2－4区，连接入pcDNA3.1载体后电泳及测序鉴定；以羧甲基化 葡聚糖纳米磁颗粒为基因载体分别携带两种重组质粒，转染人脐静脉血 管内皮细胞（HUVEC）后分别进行正常、缺氧和高糖条件下的培养。MTT 法检测各组HUVEC的增殖；光学低倍镜下观察HUVEC的迁移；分光光度计 检测兔视网膜下注射适量各组细胞培养上清液对后视网膜血管对 Evan’s blue染料的渗漏量；Western blot法检测各组细胞ERK1/2和 PI3K/Akt信号通路下游特异性磷酸化蛋白 p-ERK1/2、p-Akt的表达水平。 结果 经过pcDNA3.1/sFlt-1(2-3)或pcDNA3.1/sFlt-1(2-4)转染的 HUVEC在正常条件下的增殖、迁移及p-ERK1/2 mRNA和蛋白的表达较转染 前没有明显改变（p0.05），而经过两种重组质粒转染的HUVEC在缺氧和 高糖条件下的增殖、迁移及p-ERK1/2 mRNA和蛋白的表达较转染前均明显 下降(p0.01)；两种重组质粒对缺氧和高糖条件下HUVEC增殖、迁移、视 网膜血管通透性的改变及ERK1/2、PI3K/Akt信号通路的影响无显著性差 异（p0.05）。 结论 sFlt-1受体胞外第2-3区和第2-4区均可抑制缺氧和高糖条件 下HUVEC的增殖、迁移、视网膜血管通透性改变及p44/p42MAPK、PI3K/Akt 信号通路，且两种受体基因片段的作用无明显差异。 关键词 糖尿病性视网膜病变；可溶性Flt-1受体；基因转染；增殖； 迁移；p44/p42MAPK；PI3K/Akt。 Impact of sFlt-1 gene fragments transfer to HUVEC on the ERK1/2 and PI3K/Akt pathways of VEGF in vitro ABSTRACT Purpose: To investigate whether various sFlt-1 gene fragments affect the biological functions, ERK1/2 and PI3K/Akt pathways of vascular endothelial growth factor (VEGF) under conditions of hypoxia or in the presence of high glucose concentrations in vitro. Methods: Plasmids expressing loops 2–3 and loops 2–4 of sFlt-1 were packed in carboxymethylated dextran-coated (CMD) nanoparticles and transferred into human umbilical vein endothelial cells (HUVECs), which were then cultured under hypoxia or in a high-glucose environment. The proliferation and migration of HUVECs were examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and low-power microscopy, respectively. Transfection-induced changes in retinal vasopermeability were observed by injecting supernatant into the eyes of rabbits. Western blot analyses were