两种fins-样络氨酸激酶受体-1基因片段对磷脂酰肌醇-3激酶／蛋白酶B之间信号通路的影响

Effects of two soluble fms-like tyrosine kinase recptor-1 gene on Phosphatidyl inositol - 3 kinase/Akt pathway

目的 观察可溶性fms-样络氨酸激酶受体-1（sFlt-1）基因胞外第2～3区和2～4区对缺氧、高糖环境下视网膜血管通透性及磷脂酰肌醇-3激酶/蛋白酶B（PI3K/Akt）之间信号通路的影响.方法 构建真核表达质粒编码增强型绿色荧光蛋白质粒（pcDNA3.1-EGFP）/s Flt-1（2～3）和pcDNA3.1-EGFP/s Flt-1（2～4）,采用酶切、电泳及基因测序鉴定.将实验分为正常对照组、缺氧组和高糖组进行.以羧甲基化匍聚糖（CMD）纳米磁颗粒为基因载体,分别携带两种重组质粒转染缺氧、高糖条件下培养的人脐静脉血管内皮细胞（HUVEC）.采用分光光度计检测各组兔视网膜血管对伊凡思蓝（EB）染料的渗漏量.逆转录聚合酶链反应（RT-PCR）和免疫蛋白印迹（Western blot）法分别检测各组细胞PI3K/Akt信号通路下游特异性磷酸化激酶Akt（p-Akt）mRNA和蛋白的表达.结果 缺氧组和高糖组注射转染细胞上清液后,视网膜血管EB渗漏量较正常对照组明显增加（t=2.476,2.515;P值均＜0.01）.缺氧组和高糖组转染了pcDNA3.1-EGFP/sFlt-1（2～3）和pcDNA3.1-EGFP/sFlt-1（2～4）后,视网膜血管EB渗漏量较未转染明显降低（t=2.598,2.679,2.386,2.732;P值均＜＜0.01）.缺氧组、高糖组HUVEC p-Akt mRNA表达较正常对照组明显上调（t=2.612,2.545;P值均＜0.01）,转染pcDNA3.1-EGFP/sFlt-1（2～3）和pcDNA3.1-EGFP/sFlt-1（2～4）后明显下调（t=2.512,2.189,2.372,2.687;P值均＜0.01）.缺氧组和高糖组HUVEC p-Akt蛋白表达较正常对照组明显上调（t=2.376,2.712;P值均＜0.01）,转染pcDNA3.1-EGFP/sFlt-1（2～3）和pcDNA3.1-EGFP/sFlt-1（2～4）后明显下调（t=2.259,2.391,2.399,2.413;P值均＜0.01）.结论 sFlt-1受体胞外第2～3区和2～4区均可抑制缺氧、高糖环境下的视网膜血管通透性增高和PI3K/Akt通路的信号传导.

Objective To explore and evaluate the protective effects of soluble fms-like tyrosine kinase recptor-1 （sFlt-1） gene 2-3 and 2-4 transcellular region on retinal vascular leakage and phosphatidyl inositol-3 kinase （PI3K）/Akt pathway under hypoxia and/or hyperglycemia. Methods The plasmids pcDNA3. 1-EGFP/sFlt-1（2-3） and pcDNA3. 1-EGFP/sFlt-1 （2-4） were constructed.. Two of these plasmids with carboxy mcthylation glucan （CMD） magnetic particles were transfected into human umbilical vein endothelial cells （HUVEC）, which were cultured under hypoxia and/or hyperglycemia. The blood retinal barrier was evaluated by Evans blue permeation （EBP）. Then the expression of p-Akt mRNA and protein were detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot separately. Results In the diabetic rabbits, The blood retinal barrier breakdown was detected by the retinal vascular leakage of EBP.The expression of p-Akt mRNA and protein in hypoxia and hyperglycemia groups were also obviously increased. These changes were largely prevented by transfection the plasmids pcDNA3.1-EGFP/sFlt-1（2-3） and pcDNA3.1-EGFP/sFlt-1（2-4） （P〈0.01 in both groups）. Conclusion Both sFlt-1（2-3） and sFlt-1（2-4） can make the retinal blood vessels less leaky and may be beneficial in preventing vision loss from diabetic retinopathy.