【作者】 于品；

【导师】 秦川；

【作者基本信息】 北京协和医学院 ， 病理学与病理生理学， 2014， 博士

【摘要】 肠道病毒71型(enterovirus71,EV71)是引起手足口病(hand,foot, and mouth disease,HFMD)和导致严重神经系统并发症的常见病原体,主要流行于亚太地区。婴幼儿感染EV71引起的HFMD可能导致严重并发症,包括心肌炎、急性弛缓性麻痹、无菌性脑膜炎、脑干脑炎、神经源性肺水肿甚至死亡。然而重症EV71感染致病机制仍未明确阐明。目的本研究目的是通过病毒和宿主两方面研究EV71病毒导致患者罹患重症机制。主要通过对轻症和重症小鼠模型的建立以及比较研究,从实验动物模型方面对重症发病机制进行探讨；通过对轻症和重症病人血清差异蛋白分析,探索与重症转归相关的早期生物学因子；以及通过重症死亡患者和小鼠模型的比较病理学研究揭示重症神经性疾病和并发症的发病机制；从肠道益生菌免疫调节的角度出发,初探干酪乳杆菌在EV71感染中的抗损伤和抗感染作用,为进一步探讨黏膜免疫等方面的调节作用提供理论依据。方法1、通过体外实验比较9株临床病人分离株在神经细胞系中复制能力的差异,并对复制力有差异的病毒株进行全基因组序列分析。2、另外,分别建立小鼠轻症、重症模型,并在小鼠模型上对EV71病毒复制力、抗原在组织中的分布、血清中细胞因子浓度差异进行比较。3、对EV71病毒感染小鼠模型和EV71重症患者开展了比较病理学研究,研究内容包括EV71抗原在组织中分布,受体(SCARB2)分布定位以及血脑屏障基本结构变化,以及原位杂交定位组织中的EV71病毒RNA,并对炎性细胞的分类和分布进行了检测。4、对采集到的临床轻症、重症手足口病患者样本进行了比较、分析,并进一步用iTRAQ技术分析轻症和重症患者血清中表达差异蛋白,为找到重症预警标志物提供数据支撑。5、采用重症EV71小鼠模型对干酪乳杆菌的抗感染作用进行初探,对组织病变和病毒分布等进行检测分析,为探讨干酪乳杆菌在其中的调节机制提供实验依据。结果1、研究结果显示9株EV71毒株都属于C4基因亚型,都能在人源神经细胞系中复制。2、轻症和重症小鼠模型临床表现为,重症组小鼠存活率为71.43%,60%的小鼠出现后肢瘫痪,轻症组小鼠表现竖毛外无其他异常反应。血清中细胞因子检查发现,重症组感染初期IL-5(6hpi)、IL-13(2dpi)、IL-6、MCP-1、 CCL5/RANTES(3dpi)表现明显上调,感染后期TNF-α(7dpi)表现明显上调。3、比较病理学研究表明EV71感染死亡患者出现严重的脑脊髓炎、肺水肿和坏死性肠炎,EV71病毒和病毒受体SCARB2主要分布子中枢神经系统(central nervous system,CNS)神经元、小胶质细胞和炎性细胞以及肠上皮细胞。原位杂交结果显示CNS、肠道、肺和扁桃体浸润的炎性细胞中EV71病毒RNA反应阳性,病理检查可发现CNS中典型的血管袖套、胶质结节、嗜神经现象。重症EV71病毒性脑脊髓炎患者的血脑屏障被严重破坏。重症小鼠模型临床表现后肢瘫痪,是以大面积坏死性肌炎为主要病理表现,全身性肌坏死最终导致呼吸衰竭等并发症,致使CNS出现不同程度病变,脊髓前角运动神经元损伤,间质性肺炎等病变。4、通过比较轻症和重症患者临床数据、样本发现,体温、血糖、中性粒细胞比例、CK-MB和LDH血清水平升高是重症的危险因素,可能是与患者体内过强的免疫反应有关。采用iTRAQ技术和通路分析表明轻症和重症患者差异蛋白主要参与免疫应答和细胞凋亡。5、干酪乳杆菌可以降低EV71感染小鼠组织中的病毒载量,减轻组织炎性反应,肠道局部黏膜病变改善,可能是干酪乳杆菌增强局部黏膜免疫,从而使肠道、肺脏等组织中的病毒载量减少,提高小鼠存活率,有显著预防EV71病毒感染的作用,同时有一定抗EV71病毒感染作用。结论EV71病毒对人具有神经嗜性,而对小鼠具有肌嗜性。重症小鼠模型血清上调的细胞因子,包括IL-5、IL-13、IL-6、MCP-1和CCL5/RANTES,可能是潜在的提示重症预后的标志物。EV71死亡患者病理特征为严重的神经损伤伴有炎性细胞反应,大量浸润的巨噬细胞/小胶质细胞和中性粒细胞可能与EV71复制、CNS传播、神经损伤和并发症有关；EV71小鼠模型病理表现主要是坏死性肌炎,是重症小鼠模型的典型特征,CNS病变尤其是脊髓损伤与肢体瘫痪有一定的关联性。过强免疫反应可能导致EV71感染的HFMD患者发生重症并发症甚至出现死亡。干酪乳杆菌可能通过增强黏膜免疫等作用抗EV71病毒感染,而其具体调节机制有待进一步深入研究。本研究通过对EV71病毒感染力、轻症和重症小鼠模型比较研究、死亡患者和小鼠模型比较病理学研究以及对轻症和重症患者血清差异蛋白的研究,为阐明EV71病毒感染导致的重症神经致病机理、诊断和治疗提供理论依据和数据支撑,并为EV71动物模型建立提供疾病模型的基础研究资料。更多还原

【Abstract】 Enterovirus71(EV71) is a common pathogen that causes hand, foot, and mouth disease (HFMD) and severe neurological complications mainly in the Asia-Pacific region. HFMD associated with EV71infection in young children may cause severe complications, such as myocarditis, acute flaccid paralysis, aseptic meningitis, brainstem encephalitis, neurogenic pulmonary edema and even death. However, the mechanism of severe EV71infection remains unclear.Objectives The objective of this study was to investigate the mechanism of severe EV71infection through virus and host, respectively. By the establishment and comparative study of severe and mild mouse models of EV71infection, our research explores the severe pathogenesis from the aspects of experimental animal models. In order to explore the early biological factors associated with severe outcome, analysis of the differencial expressed proteins in sera of mild and severe patients was performed. Through the pathological comparison of fatal patients and mouse model, our study may improve the mechanistic understanding of the pathogenesis of the severe neurological diseases and complications. From the perspective of the immunoregulation of intestinal probiotics, we explore the anti-injury and anti-infective effects of Lactobacillus casei on EV71infection, which may provide basic information for further expounding the mucosal immunoregulation.Methods1. In vitro experimentations were carried out for comparison of replication and complete genome sequencing of nine EV71clinical isolates.2. Severe and mild mouse models of EV71infection were established to compare the pathological characteristics, viral replication in tissues, as well as the proinflammatory cytokine production in sera.3. To understand the pathogenesis of severe EV71-associated disease, we carried out pathological detections for locating EV71antigen and receptor (SCARB2) as well as the basic structural changes of blood-brain barrier (BBB) in human cases and mouse model. We conducted in situ hybridization for detecting EV71viral RNA in tissues of fatal patients, and meanwhile, we elucidated immunopathological mechanism in EV71infection via classification and distribution of inflammatory cells.4. We adopted clinical index statistics and iTRAQ analysis to determine biomarker candidates for a risk assessment for severe development in sera of patients with severe or mild HFMD.5. We investigated the anti-infective effects of Lactobacillus casei on severe EV71infection in mice, and the tissue lesions and virus distribution were detected, which may provide basic experimental data for further explaining the regulatory mechanism of Lactobacillus casei in EV71infection.Results1. Nine EV71isolates, which belong to subgenogroup C4, showed infectivity to SK-N-SH cells, suggesting strong neurotropism.2. Severe and mild EV71mouse models were firstly established, and the survival rate of severe model was71.43%, and60%of the surviving mice of severe model had sequelae of paralysis while the mice of mild model showed ruffled fur. Dynamic detection of serum cytokines and chemokines showed that IL-5(6hpi), IL-13(2dpi), IL-6, MCP-1, CCL5/RANTES (3dpi) were significantly up-regulated at the early stage of severe EV71infection, suggesting that these factors might herald a severe outcome.3. Microscopically, human fatal cases exhibited severe encephalomyelitis, pulmonary edema and necrotic enteritis. In human cases, EV71antigen and SCARB2were observed mainly in the neurons, microglia cells and inflammatory cells in the central nervous system (CNS), epithelial cells in the intestines. EV71RNA was mainly detected in inflammatory cells located in the CNS, intestines, lungs and tonsils. Morphologically, most of EV71RNA-positive inflammatory cells in CNS were macrophages/microglia and neutrophils infiltrated in perivascular cuffing, microglial nodule, neuronophagia and meninge. Moreover, the BBB in patients with EV71viral encephalomyelitis was severely disrupted. Comparatively, in our mouse model, we observed massive necrotic myositis, different degrees of viral diseases in CNS, lesions in anterior motor neurons of the spinal cord, extensive interstitial pneumonia. And the systemic necrotic myositis may contribute to the respiratory failure and other complications.4. We identified that high level of body temperature, blood sugar, neutrophilic granulocyte ratio, CK-MB and LDH, which might be highly related to the overactive immune response. Using iTRAQ-method and pathways analysis, we found that differentially expressed proteins in sera of severe and mild patients are mainly involved in the immune response and cell death related process.5. Administration of Lactobacillus casei could reduce the viral loads and pathological changes, ameliorate the focal lesions in the mucous membrane of the intestines. The mucosal immune may be up-regulated by Lactobacillus casei, and the virus loads in the intestines and lungs were then reduced, which improve survival rate of the EV71infection in mice. Our data suggest that Lactobacillus casei showed protective effect and anti-infection to EV71infection.Conclusions EV71strains commonly possess neurotropism to humans. The elevated cytokines and chemokines, including IL-5, IL-13, IL-6, MCP-1and CCL5/RANTES, may have potential value as severe prognostic markers in severe mouse model. Fatal patients of EV71infection are characterized as severe neurological lesions with intense inflammatory cells reaction. Massively infiltrated macrophages/microglia and neutrophils might be associated with EV71replication, transmission to CNS, neurological impairment, and neurological complications. EV71mouse model mainly exhibits necrotizing inflammation in skeletal muscle, which is the typical characteristics of severe mouse model. And changes in the CNS, especially in anterior motor neurons may have certain association with paralysis in limbs. Overactive immune response induced by EV71infection may lead to severe and fatal prognosis in HFMD patients. Lactobacillus casei may enhance the mucosal immune to play a role in the anti-infective effect against EV71infection, but the specific regulatory mechanism remains to be furthur study. Our study may provide valuable theoretical foundation and disease models for understanding neurologic pathogenesis of fatal EV71prognosis, and diagnostic and therapeutic values for EV71infection.更多还原