登革病毒感染诱导肠黏膜屏障功能损伤的初步研究  

Primary study on functional injuries of intestinal mucosal barrier induced by Dengue virus

作　　者：陈炜 徐小峰 田衍平 王嘉丽 张俊磊 CHEN Wei;XU Xiao-feng;TIAN Yan-ping;WANG Jia-li;ZHANG Jun-lei(The Second Affiliated Hospital,Army Medical University,Chongqing,400037,China)

机构地区：[1]陆军军医大学第二附属医院感染控制科,重庆400037 [2]陆军军医大学基础医学院医学遗传学教研室,重庆400038 [3]陆军军医大学基础医学院组织胚胎学教研室,重庆400038

出　　处：《中华医院感染学杂志》2019年第20期3050-3055,共6页Chinese Journal of Nosocomiology

基　　金：第三军医大学基础医学前沿基金资助项目(2012XJQ05)

摘　　要：目的对登革病毒感染诱导肠黏膜屏障功能损伤进行初步的研究,为阐明登革出血热/登革休克综合征(DHF/DSS)的发病机制提供理论依据。方法采用登革2型病毒(Dengue virus 2,DV2)感染RhoA突变体稳定表达细胞株,运用病毒噬斑计数法观察其对DV感染的影响。基于体外HMEC-CaCO2细胞共培养模型,观察DV感染导致肠上皮细胞屏障功能损伤和紧密连接蛋白分布和网络结构的变化情况。运用酶联免疫吸附测定法(Enzyme-linked immunosorbent assay,ELISA)和聚合酶链式反应(Polymerase chain reaction,PCR)法观察在DV感染条件下协同脂多糖(Lipopolysaccharide,LPS)刺激对血管内皮细胞分泌白细胞介素-8(Interleukin-8,IL-8)和IL-6的影响。结果干扰RhoA/ROCK的活性会抑制DV的复制增殖,ECVWtRhoA、ECVV14RhoA和ECVN19RhoA培养上清和细胞内的病毒滴度均低于ECV304N,分别下降了88.40%、51.17%、25.33%和35.83%、15.21%、10.63%。HMEC感染DV后与CaCO2细胞共培养1天后与对照组相比跨上皮电阻(Trans-epithelial resistance,TER)下调了(27.00±4.55)Ω/cm2(P=0.002),2天后跨上皮电阻下调了(40.33±0.12)Ω/cm2(P<0.0001)。同时,Ca-CO2细胞紧密连接蛋白ZO-1、occludin的分布发生改变,紧密连接网络受到破坏,肠上皮细胞层通透性增高。此外,在DV感染条件下协同LPS刺激与单独感染组相比可进一步促进血管内皮细胞分泌IL-8和IL-6(P<0.001)。结论DV感染血管内皮细胞可能在RhoA/ROCK通路活化和其他因素的共同参与下诱发肠黏膜屏障功能损伤和肠道细菌移位,由此启动的LPS对血管内皮的持续刺激可能成为血管内皮损伤和血浆渗漏发生的“加速器”,推动DHF/DSS病程发展,但其机制仍需进一步证实。OBJECTIVE To explore the functional injuries of intestinal epithelial barrier(IEB)induced by dengue virus(DV)infection,so as to provide basic evidence for elucidating the mechanism of dengue hemorrhagic fever(DHF)and dengue shock syndrome(DSS).METHODS Stable expression cell lines of RhoA mutant were infected with Dengue virus 2(DV2),and the infectious effect were determined by viurs plaque assay.Based on the HMECCaCO2co-culture system,the IEB functional injuries resulted from DV infection and distribution and network structure of tight junction protein were observed.With DV infection,the secretion of IL-6and IL-8by vascular epithelial cells,synergized with lipopolysaccharide(LPS)stimulation,were determined by enzyme-linked immunosorbent assay(ELISA)and polymerase chain reaction(PCR).RESULTS Interference of RhoA/ROCK pathway activity significantly restrained the proliferation of DENV2.Compared with the ECV304Ncontrol,the titers of intracellular and supernatant of ECVWtRhoA,ECVV14RhoAand ECVN19RhoAgroups were significantly reduced by 88.40%,51.17%,25.33%or 35.83%,15.21%,10.63%respectively.Co-cultureed DV-infected HMEC with CaCO2,transepithelial resistance(TER)was reduced by(27.00±4.55)Ω/cm2 and(40.33±0.12)Ω/cm2,respectively,compared to mock infection group at 24hor 48hpostinfection(p.i),accompanied with distribution alternation of tight junction protein ZO-1and Occludin,damage of tightly connected webwork,and increased permeability of intestinal epithelial.In addition,under DV infection,compared with single infection group,synergized stimulation of LPS may further promote IL-8and IL-6secretion of vascular epithelial cells(P<0.001).CONCLUSION The dysfuction of intestinal epithelial barrier and bactrial translocation might be induced by cooperated participating of RhoA/ROCK pathway and other factors of DV-infected vascular epithelial cells,thereby initiating continuous stimulation on the vascular epithilial cells by LPS,which might be the accelerator of vascular epithelial injury and plasam leakage and drive the

关 键 词：登革病毒 登革出血热/登革休克综合征 肠黏膜屏障 RhoA/ROCK 内毒素 白细胞介素-6 白细胞介素-8 

分 类 号：R373.33[医药卫生—病原生物学]