臺灣博碩士論文加值系統

腸病毒七十一型主要感染對象為嬰兒及幼童，在臨床上病毒感染所造成的症狀有：手口足病、疱疹性咽峽炎、無菌性腦膜炎、腦炎、肢體麻痺，甚至死亡。但是目前對於腸病毒感染造成的疾病輕重程度與病人體內病毒數量的關係都還不太清楚。因此本研究首先藉著運用定量反轉錄-聚合酵素連鎖反應(QR-PCR)的建立來偵測腸病毒病人檢體中之病毒量。所利用的引子對腸病毒七十一型具有高度專一性，且所建立的腸病毒分析的敏感度可達每毫升檢體約可偵測103至106個正負股病毒基因組。我們收集並分析了20位病人的周邊單核血球細胞，但是卻偵測不到病毒的存在。除此外我們在老鼠上建立了餵食及腹腔注射感染的動物模式，發現不只是老鼠年齡影響了感染後存活率的多寡，而且老鼠的體重輕重也是存活率的因子之一。腸病毒在老鼠會造成全身性感染，病毒量又以神經性器官為最多。老鼠被感染的內臟，也有許多免疫細胞浸潤，例如嗜中性白血球，在一些病毒相關研究中指出，嗜中性白血球在一些好神經性病毒的感染中有著舉足輕重的角色，因此本實驗欲探討嗜中性白血球在腸病毒七十一型感染中所扮演的角色。所以接著當我們在餵食感染老鼠前後以嗜中性白血球專一抗體RB6-8C5來清除老鼠體內的嗜中性白血球後，發現老鼠的存活率降低了百分之五十五。在感染後7天後，在嗜中性白血球被清除且被病毒感染的老鼠中，腦及脊髓中病毒的量明顯的比只有被病毒感染的老鼠中來的高。研究結果顯示在餵食感染腸病毒七十一型的動物模式下，嗜中性白血球在避免老鼠感染後死亡及抑制病毒生長上有著相當重要的角色。

Enterovirus 71 (EV71) infection causes many clinical syndromes, including hand-foot-mouth disease, herpangina, aseptic meningitis, encephalitis, and paralysis, particularly, in infants and children. The relationship between disease progression and virus load is unclear. To investigate the virus load in serum samples collected from EV71 patients, I developed a reverse-transcription PCR assays to quantify EV71 genome in samples. These assays are specific for EV71. The detection limit is 103 copies genome per milliliter serum sample for both positive- and negative-stranded genome assays. When these assays were used to determine the amount of virus in peripheral blood mononuclear cells from 20 patients, no viruses can be detected in these samples. A mouse model was also established to investigate the pathogenesis of EV71 infection of intragastral (i.g.) or intraperitoneal (i.p.) inoculation. Results showed that the mouse survival rate was dependent on not only the age but also the body weight of mice. In EV71 infected-mice, viruses caused a systemic infection and their titers in neuronal organs were higher than those in non-neuronal organs during late infection. Immune cells, such as neutrophils, infiltrated into infected organs. Many reports indicated that neutrophils play an important in neurotropic virus infections. To investigate the role of neutrophils in EV71 infection, monoclonal antibody specific for granulocytes, RB6-8C5, was used to deplete neutrophils before and after oral infection with EV71. I found neutrophil depletion decreased mouse survival. The virus titer in the brain and spinal cord of neutrophil depleted mice was significantly higher than those of normal control mice at day 7 post infection. This result suggested that neutrophils play an important role in protecting mice from death and inhibiting virus growth after EV71 oral infection.

Chinese abstract i
English abstract iii
Acknowledgement v
Contents vi
Introduction 1
Material and methods 6
Virus and cells 6
Preparation of total RNA from patient PBMC and virus-infected cells 6
Quantitative reverse-transcription polymerase chain reaction (QR-PCR) 7
Infection of mice and tissue collection 8
Antibody purification 8
Neutrophil depletion during EV71 infection 9
Immunohistochemistry (IHC) staining 10
Histological examination 10
Cocultivation of EV71 and neutrophils in vitro 10
Statistical analysis 11
Results 12
Determination of primers specificity 12
Establishment of RNA standards for QR-PCR 12
Analyses of PBMC from EV71 patients 13
Establishment of a mouse model for EV71 infection 13
The effect of neutrophil depletion on EV71-infected mice 16
Interaction of neutrophils and virus in vitro 17
Discussion 19
Detection of viruses in patient PBMC samples 19
Establishment of mouse model foe EV71 infection 20
The role of neutrophils in EV71 infection 21
References 23
Table 31
Figures 32

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