摘要
目的探讨氧糖剥夺再灌(OGD/R)条件下,小鼠脑内皮细胞bEnd.3中微小RNA-290b-3p(miR-290b-3p)在体外血脑屏障模型的作用与机制。方法培养小鼠脑内皮细胞bEnd.3,建立OGD/R模型与体外血脑屏障模型,Transwell法检测氧糖剥夺后体外血脑屏障模型通透性的变化;实时荧光定量聚合酶链式反应(real time-PCR,RT-PCR)检测OGD/R后miR-290b-3p表达水平;Targetscan网站预测miR-290b-3p与紧密连接蛋白5(Claudin-5)结合;荧光素酶报告实验验证miR-290b-3p与Claudin-5结合;转染bEnd.3细胞miR-290b-3p抑制剂,应用蛋白质印迹法(Western Blot)检测Claudin-5的表达,Transwell法检测抑制miR-290b-3p后氧糖剥夺后体外血脑屏障模型通透性变化。结果OGD/R后体外血脑屏障破坏,1、2、3、4、6 h荧光素异硫氰酸酯(FITC)-葡聚糖扩散速率分别为(0.04±0.00)、(0.04±0.01)、(0.05±0.01)、(0.07±0.01)、(0.12±0.01)pmol·mm^-2·min^-1,通透性增加;miR-290b-3p在OGD/R后1、2、3、4、6 h表达量分别为(1.18±0.12)、(1.27±0.12)、(1.55±0.18)、(2.13±0.18)、(2.53±0.15),表达增加;miR-290b-3p与Claudin-5结合;抑制miR-290b-3p可以缓解OGD/R后Claudin-5的降低,减轻血脑屏障的损伤。结论氧糖剥夺后bEnd3细胞中miR-290b-3p表达增加抑制miR-290b-3p可通过调节Claudin-5表达减轻氧糖剥夺模型中血脑屏障的损伤。
Objective To explore the role of miR-290 b-3 p in the blood-brain barrier model in vitro under oxygen-glucose deprivation and reperfusion conditions.Methods Mouse brain endothelial cells bEnd.3 were cultured,oxygen glucose deprivation reperfusion model(OGD/R)and blood-brain barrier model in vitro were established,Transwell method was used to detect the permeability of bloodbrain barrier model in vitro after oxygen glucose deprivation,and miR-290 b-3 p expression level after OGD/R was detected by real timePCR.Targetscan algorithm was used to predict the interaction of miR-124 and Claudin-5;the binding of miR-290 b-3 p and Claudin-5 was detected by Luciferase;transfection of miR-290 b-3 p inhibitor in bEnd3 cells and detection of Claudin-5 expression by Western Blot and permeability of blood-brain barrier model in vitro.Results After OGD/R,the blood-brain barrier was destroyed,the diffusion rates of fluorescein isothiocyanate(FITC)-dextran were(0.04±0.00),(0.04±0.01),(0.05±0.01),(0.07±0.01),(0.12±0.01)pmol·mm^-2·min^-1.at 1,2,3,4,and 6 h,respectively,and permeability increased.The expression of miR-290 b-3 p increased after OGD/R[1,2,3,4,6 h:(1.18±0.12),(1.27±0.12),(1.55±0.18),(2.13±0.18),(2.53±0.15)];miR-290 b-3 p combined with Claudin-5;inhibition of miR-290 b-3 p reversed decrease of Claudin-5 and alleviated the damage of the blood-brain barrier during OGD/R.Conclusion The expression of miR-290 b-3 p is increased induced by OGD/R in bEnd3,inhibition of miR-290 b-3 p attenuates blood-brain barrier damage in the oxygen-sugar deprivation model by modulating Claudin-5 expression.
作者
张奕雯
凡子莲
李经伦
熊兰
赵自胜
李超
ZHANG Yiwen;FAN Zilian;LI Jinglun;XIONG Lan;ZHAO Zisheng;LI Chao(Department of Neurology,The First People's Hospital of Guangyuan,Guangyuan,Sichuan 628000,China;Department of Radiology,The First People's Hospital of Guangyuan,Guangyuan,Sichuan 628000,China;Department of Neurology,The Affiliated Hospital of Southwest Medical University,Luzhou,Sichuan 646000,China)
出处
《安徽医药》
CAS
2021年第2期345-348,共4页
Anhui Medical and Pharmaceutical Journal
