VS-1基因转染对血管内皮细胞缺氧/复氧损伤的保护作用

Protection of VS-1 transgenic therapy against HUVEC hypoxia/reoxygenation injury

目的构建Ad-VS-1基因转染HUVEC的体外缺氧/复氧模型,探讨VS-1转基因治疗对血管内皮细胞缺氧/复氧损伤的保护作用及其机制。方法①建立HUVEC缺氧/复氧(H/R)损伤模型,分为4组:空白组,H/R组,空载腺病毒转染+H/R组,Ad-VS-1转染+H/R组;②测定内皮细胞活力(MTT),超氧化物歧化酶活力(SOD)、丙二醛(MDA)、eNOS、NO表达、炎症因子ICAM-1、VCAM-1、TNF-α的表达情况;③在Ad-VS-1转染+H/R组基础上增设Hb、KT5823、SB203580和W ortm an in 4个信号抑制组,流式细胞仪测定细胞凋亡率进行统计学分析。结果①成功构建Ad-VS-1转染HUVEC表达后H/R模型;②与空白组比较,H/R组和空腺病毒感染+H/R组的MTT、SOD含量、eNOS、NO表达显著降低,而MDA、ICAM-1、VCAM-1、TNF-α含量显著增加(P<0.05);与H/R组和空载腺病毒感染+H/R组比较,VS-1转染+H/R组MTT、SOD、eNOS、NO表达含量明显回升,而MDA、ICAM-1,TNF-α生成量明显回落(P<0.05);③与H/R组和空载腺病毒感染+H/R组比较,VS-1转染组细胞凋亡率明显降低,而4组信号抑制剂组的细胞凋亡率较VS-1转染组均明显回升(P<0.05)。结论Ad-VS-1成功转染HUVEC,并通过PI3K/Akt-eNOS-NO-cGMP-PKG和P38MPAK等信号途径,抑制氧化应激和炎症反应,产生显著的抗血管内皮细胞缺氧/复氧损伤作用,为今后VS-1抗心肌缺血/再灌注损伤研究奠定基础。

Objective To study the protective effects and mechanisms of VS-1 transgenic therapy on vascular endothelial hypoxia/reoxygenation after construction of hypoxia/reoxygenation （H/R） model injury by transfecting Ad-VS-1 gene in human umbilical vein endothelial cells （HUVEC） in vitro. Methods （1） HUVEC H/R models were established and divided into 4 groups ： control group, H/R group, blank adenovirus infection ＋ H/R group and Ad-VS-1 infection ＋ H/R group.（2）Endothelial viability was assayed by MTT and superoxide dismutase enzyme （SOD）, and the expression of malondialdehyde （MDA）, endothelial nitric oxide synthase （eNOS）, nitric oxide （NO） and inflammatory factors such as intercellular adhesion molecule-1 （ICAM-1）, vascular cell adhesion molecule-1 （VCAM-1） and tumor necrosis factor-α （TNF-α） were detected by respective routine methods. （3）Hb, KT5823, SB203580 and Wortmanin inhibitors were added respectively into Ad-VS-1 infection ＋ H/R group, and apoptosis rate were measured by flow cytometry in all groups. Results （1）H/R model were successfully constructed by transfecting Ad-VS-1 gene. （2） Compared with control group, expressions of MTT, SOD, eNOS and NO in H/R group and blank-Ad infection ＋ H/R group were significantly reduced while MDA, ICAM-1, VCAM-1 and TNF-α concentration increased significantly （P 〈 0.05）. Compared with H/R group and blank-Ad infection ＋ H/R group, expressions of MTT, SOD, eNOS and NO in Ad-VS-1 infection ＋ H/R group reversed, while expressions of MDA, ICAM-1 and TNF-ot decreased （P 〈 0.05）. （3） Compared with H/R group and blank-Ad infection ＋ H/R group, apoptosis rate in Ad-VS-1 infection ＋ H/R group decreased significantly while apoptosis rates in 4 signal inhibitor groups all increased more than that in Ad-VS-1 infection ＋ H/R group （ P 〈 0.05 ）. Conclusion Ad-VS-1 transfection and H/R model of HUVEC has been constructed successfully which inhibits the oxidative stress and inflammation reaction through the PI3K/Akt-eNOS-NO-cGMP-PKG and P38MPAK signaling pathways. VS-1 transgenic therapy exerts significant protective effects against H/R injury in vascular endothelial cells, laying most solid foundation for further research of myocardial protective effect of VS-1.