靶向人Gadd45α基因的shRNA慢病毒载体对缺氧致人脐静脉内皮细胞生物学功能损伤的影响

Effect of shRNA Lentiviral Vector Targeting Human Growth Arrest and DNA Damage 45α Gene on Disfunction of Human Umbilical Vein Endothelial Cells Due to Hypoxia Stress

目的研究靶向人生长阻滞和DNA损伤45α(Growth arrest and DNA damage 45 alpha,Gadd45α)基因和表达绿色荧光蛋白(Green fluorescert protein,GFP)的shRNA慢病毒载体对缺氧致人脐静脉内皮细胞(Human umbilical vein endothelialcells,HUVECs)表达上调的Gadd45α的沉默作用,初步阐明Gadd45α在缺氧应激致HUVECs生物学功能损伤过程中的作用。方法慢病毒包装后感染HUVECs,筛选最适MOI及感染时间。感染72h后,采用Real-time PCR和Western blot检测细胞中Gadd45αmRNA和蛋白的表达;流式细胞术检测细胞的凋亡率;Transwell小室实验检测细胞的迁移率;ELISA检测细胞sFlt-1和sEng的分泌水平。结果包装后慢病毒载体的滴度为1×108TU/ml,最适MOI为20,最适感染时间为72h,对HUVECs的感染效率约为80%。Gadd45α shRNA慢病毒载体对Gadd45α基因的沉默效率可达80%,阴性对照慢病毒载体对Gadd45α的表达无抑制作用。缺氧可致HUVECs中Gadd45α的表达上调,靶向Gadd45α基因的shRNA慢病毒颗粒可有效抑制缺氧致HUVECs的凋亡,减少sFlt-1及sEng的释放,同时增强其体外迁移能力,与缺氧组相比,差异具有统计学意义(P<0.05)。Gadd45α蛋白的表达水平与sFlt-1及sEng的分泌水平呈正相关(r1=0.89,r2=0.77,P均<0.05)。结论沉默Gadd45α基因对缺氧应激条件下的HUVECs生物学功能具有保护作用;Gadd45α可能是一个关键上游位点,参与子痫前期时sFlt-1及sEng的释放。

Objective To study the silencing effect of shRNA lentiviral vactor,targeting human growth arrest and DNA damage 45α（Gadd45α）gene and expressing green fluorescent protein（GPF）,on the up-regulation of Gadd45α expression in human umbilical vein endothelial cells（HUVECs）due to hypoxia stress,and preliminarily investigate the role of Gadd45α in disfunction of HUVECs due to hypoxia stress.Methods HUVECs were infected with the lentiviral vector after package,and the MOI and time for infection were optimized.The HUVECs were determined for expressions of Gadd45α mRNA and protein by real-time PCR and Western blot 72 h after infection,while for apoptosis rate by flow cytometry,for migration rate by Transwell test,and for secretion levels of sFlt-1 and sEng by ELISA.Results The final titer of lentiviral vector after package was 1 × 108 TU/ml,and the optimal MOI and time for infection were 20 and 72 h respectively.The infection efficacy of HUVECs was about 80%.The silencing efficacy of Gadd45α gene with Gadd45α shRNA lentiviral vector reached 80%,while the lentiviral vector as negative control showed no inhibitory effect on Gadd45α expression.Hypoxia stress caused the up-regulation of Gadd45α expression in HUVECs,however,the shRNA lentiviral vector targeting Gadd45α gene inhibited the apoptosis of HUVECs due to hypoxia stress,decreased the releases of sFlt-1 and sEng,and enhanced the migration in vitro of HUVECs（P 〈0.05）vs the HUVECs cultured under hypoxia stress and untreated with the shRNA lentiviral vector targeting Gadd45α gene.The expression level of Gadd45α protein was positively related to the secretion levels of sFlt-1 and sEng（r1 = 0.89,r2 = 0.77,both P〈 0.05）.Conclusion Silencing Gadd45α gene showed protective effect on the biological function of HUVECs under hypoxia stress,and Gadd45α might be a key upstream activation site involved in regulating the releases of sFlt-1 and sEng in preeclampsia.