汉滩病毒感染诱导血管内皮细胞多糖包被损伤及其机制初步研究

Glycocalyx injury in vascular endothelial cells induced by hantaan virus infection and its preliminary mechanism

目的探讨汉滩病毒(hantaan virus,HTNV)感染血管内皮细胞致多糖包被(glycocalyx,GCX)损伤的分子机制。方法利用HTNV感染人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs),在感染不同时间段,ELISA检测细胞上清中GCX组分(硫酸乙酰肝素、硫酸软骨素、透明质酸、可溶性CD138和磷酯酰肌醇聚糖),实时定量PCR和Western blot检测蛋白聚糖中核心蛋白(CD138、磷酯酰肌醇聚糖和CD44)及相关分解酶(乙酰肝素酶、中性粒细胞弹性蛋白酶和透明质酸酶)的表达,间接免疫荧光检测糖蛋白的分布情况,跨内皮电阻检测细胞通透性变化。结果HTNV感染HUVECs后,随着感染时间的延长,释放至细胞上清液中的粘多糖成分(硫酸乙酰肝素、硫酸软骨素、透明质酸)逐渐增多。与其相连的核心蛋白在细胞中表达亦逐渐增多,其中CD138和磷酯酰肌醇聚糖mRNA的表达水平上升,在48 h和72 h分别上调3.68倍和2.47倍(P均<0.05),蛋白表达水平也出现上升。免疫荧光检测结果显示HTNV感染后细胞上糖蛋白表达减少。同时,乙酰肝素酶和中性粒细胞弹性蛋白酶mRNA表达水平出现上升,在48 h和24 h分别上调2.23倍和2.76倍(P均<0.05)。跨内皮电阻检测结果显示,弹性蛋白酶抑制剂(1μM)预处理后跨内皮电阻值上升。结论HTNV感染可能引起血管内皮细胞GCX损伤,通过上调或活化某些脱落酶,酶解GCX,致屏障结构破坏和通透性增加,为阐明HTNV感染致血管内皮损伤的分子机制提供了重要资料。

Objective To investigate the molecular mechanism of glycocalyx(GCX)injury in vascular endothelial cells due to hantaan virus(HTNV)infection.Methods Human umbilical vascular endothelial cells(HUVECs)were infected by HTNV.At different time points of HTNV infection,ELISA was used to detect the GCX components(heparan sulfate,chondroitin sulfate,haluronic acid,sCD138 and glypican)in the cellular supernatant.Real-time quantitative PCR and Western blot assay were used to detect the expression of core proteins(CD138,glypican and CD44)in the proteoglycan and their related decomposition enzymes(heparanase,neutrophil elastase and hyaluronidase).Immunofluorescence assay was used to detect the distribution of the glycoproteins.Transendothelial electrical resistanc(TEER)was used to detect the change of cellular permeability.Results With the duration of HTNV infection in vascular endothelial cells,mucopolysaccharide components(heparan sulfate,chondroitin sulfate and haluronic acid)released into the cellular supernatant gradually increased,and the expression of the associated core proteins in cells also gradually increased.Among them,the expression levels of CD138 and glypican mRNA increased,with 3.68 times at 48 h and 2.47 times at 72 h(P<0.05),respectively,and the protein expression levels also increased.Immunofluorescence detection showed that the expression of glycoprotein on cells after HTNV infection decreased.At the same time,the expression levels of heparanase and neutrophil elastase mRNA increased,with 2.23 times at 48 h and 2.76 times at 24 h(P<0.05),respectively.TEER results showed that the TEER value increased after pretreatment with elastase inhibitor(1μM).Conclusions GCX injury in vascular endothelial cells may be caused by HTNV infection,by up-regulating or activating certain shedding enzymes,GCV is enzymolyzed,the barrier structure is destroyed and the permeability increases,which provides a key theoretical basis for elucidating the molecular mechanism of vascular endothelial damage by HTNV infection.