sFlt-1基因片段对缺氧、高糖下人脐静脉血管内皮细胞及ERK1/2信号通路的影响  

Impacts of sFlt-1 on human umbilical vein endothelial cell and ERK 1/2 pathway under hypoxia or high-glucose environment

作　　者：江丹[1,2] 吴强[1] 宋蓓雯[1] 张敏[1] 杜新华[1] 贾丽丽[1] 陆斌[1] 

机构地区：[1]上海交通大学附属第六人民医院眼科,200233 [2]上海中医药大学附属龙华医院眼科,200233

出　　处：《眼科研究》2010年第9期810-815,共6页Chinese Ophthalmic Research

基　　金：上海市登山计划项目(064119543)

摘　　要：目的比较可溶性血管内皮生成因子受体1(sFlt-1)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生、迁移及细胞外调节蛋白激酶1/2(ERK 1/2)信号转导通路的影响。方法以逆转录聚合酶链反应(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,并进行测序鉴定;以羧甲基化葡聚糖(CMD)纳米磁颗粒为基因载体分别携带2种重组质粒,转染人脐静脉内皮细胞(UVECs)后分别进行正常、缺氧和高糖条件下的培养。MTT法检测各组人UVECs的增生;光学显微镜下观察人UVECs的迁移;Western blot法检测各组人UVECs ERK1/2信号通路下游特异性磷酸化激酶ERK 1/2(p-ERK 1/2)蛋白的表达。结果经过pcDNA3.1/sFlt-1(2-3)或pcDNA3.1/sFlt-1(2-4)转染的人UVECs在缺氧和高糖条件下的增生、迁移及p-ERK 1/2蛋白的表达较转染前均明显下降(P<0.01);2种重组质粒对缺氧和高糖条件下人UVECs增生、迁移及ERK 1/2信号转导通路的影响差异均无统计学意义(P>0.05)。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人UVECs的增生、迁移及ERK1/2信号通路的转导,且2种受体基因片段的作用无明显差异。Background Diabetic retinopathy（DR） is a progressive,microvascular and inevitable complication of diabetic mellitus,in which the retinal capillaries are damaged and neovascularization in retina.The mechanism of DR progession is so complicated and unclear today.ObjectivePresent study was to compare the impacts of soluble fms-like tyrosine kinase-1（sFlt-1） on the proliferation,migration of human umbilical vein endothelial cell（UVECs） and signal transduction of extracellular signal-regulated kinases 1 or 2（ERK 1/2） pathway under the hypoxia or high-glucose condition.MethodsPlasmids pcDNA3.1/sFlt-1（2-3） and pcDNA3.1/sFlt-1（2-4） were constructed by transfecting,amplifying by RT-PCR and identifying by BamHI and SalI double-enzyme cleavage method.Human UVECs were cultured under the normal,hypoxia or high-glucose environment after transfections of the two plasmids mediated by CMD（carboxymethylated dextran） nanoparticles,respectively,and pcDNA3.1-transfected human UVECs were used as control.The proliferation and migration of human UVECs in each group were observed by MTT under the low power microscope.Western blotting assay was performed to detect the expression of p-ERK 1/2 protein.ResultsThe double-enzyme cleavage method showed that recombined plasmids pcDNA3.1/sFlt-1（2-3） and pcDNA3.1/sFlt-1（2-4） fragments were obtained with the consistent length with expected ones.The expressive rate of enhanced green fluorescent protein（EGFP） was 45% after transfected.Proliferation and migration of human UVECs cultured under the hypoxia and high-glucose environment were inhibited by pcDNA3.1/sFlt-1（2-3） or pcDNA3.1/sFlt-1（2-4）（P0.01）.Downregulation of p-ERK 1/2 protein expression was also observed after transfected（P0.01）.But there was no disparity of the impacts between the two kinds of plasmids（P0.05）.ConclusionThe second to third and second to fourth extracellular region of sFlt-1 are capable of inhibiting the proliferation and migration of human UVECs and ERK 1/2 pathway

关 键 词：糖尿病视网膜病变 可溶性血管内皮生成因子受体1 基因转染 增生 迁移 细胞外调节蛋白激酶1/2信号通路 

分 类 号：R587.2[医药卫生—内分泌] R774.1[医药卫生—内科学]