Epsin2基因沉默对小鼠内皮细胞体外血脑屏障模型的影响  

Effect of Epsin2 gene silencing on blood-brain barrier model of mouse endothelial cells in vitro

作　　者：於晓东 柴文英[1] 王永刚[1] 曹奕强 龙江[1] Yu Xiaodong;Chai Wenying;Wang Yonggang;Cao Yiqiang;Long Jiang(Department of Neurosurgery,the First Affiliated Hospital of Kunming Medical University,Kunming 650000;Renmin Hospital,Hubei University of Medicine,Shiyan 442000,China)

机构地区：[1]昆明医科大学第一附属医院神经外科,昆明650000 [2]十堰市人民医院(湖北医药学院附属人民医院)

出　　处：《神经解剖学杂志》2019年第6期657-662,共6页Chinese Journal of Neuroanatomy

基　　金：云南省科技厅-昆明医科大学应用基础研究联合专项资金项目(2015FB033)

摘　　要：目的:研究使用小干扰RNA(siRNA)特异性沉默体外血脑屏障模型中小鼠脑微血管内皮细胞系b. End3细胞中Epsin2的表达后对血脑屏障模型的功能及VEGFR2、ZO1、occludin表达的影响。方法:使用化学合成的siRNA特异性干扰b. End3细胞Epsin2的表达,免疫荧光、Western Blot检测转染后细胞Epsin2、VEGFR2、ZO1、occludin的表达。体外血脑屏障模型分为对照组(control)和Epsin2-siRNA干扰组(Epsin2-siRNA),Control组使用正常b. End3细胞建立体外血脑屏障模型,Epsin2-siRNA组使用Epsin2-siRNA转染后b. End3细胞建立体外血脑屏障模型,分别于建模24、48、72 h后进行跨内皮电阻测定,48 h辣根过氧化物酶(HRP)渗透试验测定血脑屏障模型功能。结果:转染后b. End3细胞Epsin2免疫荧光和Western Bolt结果均提示表达下降(P <0. 05)。随着Epsin2的表达下降,VEGFR2表达随之下降,ZO1、occludin表达上升(P <0. 05)。Epsin2-siRNA组TEER值与Control组比,48 h后Control组与Epsin2-siRNA组TEER均升高,但Epsin2-siRNA组升高幅度高于Control组,差异有统计学意义(P <0. 05);72 h后Epsin2-siRNA干扰组TEER进一步升高且与Control组相比差异有统计学意义(P <0. 05);共培养48 h后Epsin2-siRNA干扰组通透性明显低于Control组,且有统计学差异(P <0. 05)。结论:Epsin2的表达能够影响血脑屏障相关分子的表达,从而影响血脑屏障功能。Objective: To investigate the function of blood-brain barrier model and VEGFR2,ZO1,occludin after the expression of Epsin2 in mouse brain microvascular endothelial cells b. End3 was silenced by small interfering RNA(siRNA). Methods: Using chemically synthesized siRNA to specifically interfere with the expression of Epsin2 in b. End3 cells,immunofluorescence and Western Blot were used to detect the expression of Epsin2,VEGFR2,ZO1 and occludin of transfected cells. The in vitro blood-brain barrier model was divided into control group(Control) and Epsin2-siRNA interference group(Epsin2-siRNA). The Control group used normal b. End3 cells to establish the in vitro blood-brain barrier model,and Epsin2-siRNA interference group was transfected with siRNA. The b. End3 cells established an in vitro blood-brain barrier model,and the trans-endothelial electrical resistance test and the 48 h horse radish peroxidase(HRP) permeation test were used to determine the blood-brain barrier model function after 24,48,72 hours of modeling. Results: Epsin2 immunofluorescence and Western Blot results in b. End3 cells after transfection showed a decrease in expression(P < 0. 05). With the decrease of Epsin2 expression,the expression of VEGFR2 decreased,and the expression of ZO1 and occludin increased(P < 0. 05). The TEER value of Epsin2-siRNA interference group was compared with the control group. After 48 h,the TEER of the Control group and Epsin2-siRNA interference group increased,but the increase of Epsin2-siRNA interference group was higher than that of the Control group. The difference was statistically significant(P < 0. 05). After 72 h,the TEER in the Epsin2-siRNA interference group was further increased and the difference was statistically significant(P < 0. 05). After 48 hours of co-culture,the permeability of the Epsin2-siRNA interference group was significantly lower than that of the Control group,and there was a statistical difference(P < 0. 05). Conclusion: The expression of Epsin2 can affect the expression of blood-brain bar

关 键 词：Epsin2 VEGFR2 SIRNA 血脑屏障模型 b.End3细胞 

分 类 号：R73[医药卫生—肿瘤]