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系統識別號 U0026-0812200910200984
論文名稱(中文) 腸病毒七十一型之小鼠餵食感染模式
論文名稱(英文) An murine model of enterovirus 71 infection
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 90
學期 2
出版年 91
研究生(中文) 陳怡君
研究生(英文) Yi-Chun Chen
電子信箱 ejune_chen@yahoo.com.tw
學號 s4688107
學位類別 碩士
語文別 中文
論文頁數 57頁
口試委員 指導教授-黎煥耀
召集委員-蘇益仁
口試委員-余俊強
中文關鍵字 餵食  感染  小鼠  動物模式  腸病毒71型 
英文關鍵字 infection  murine  animal model  intragastrical injection  enterovirus 71 
學科別分類
中文摘要 腸病毒71型屬於小核醣核酸病毒(Picornaviridae),其基因體為單一正股核醣核酸(single positive strand RNA)。腸病毒71型於1973年爆發流行被鑑定出後,隨後在美國、澳洲、及東亞等地陸續發生流行,造成口手足症(hand-foot-mouth disease; HFMD)等症狀;在1998年台灣的大爆發中,腸病毒71型感染伴隨嚴重的中樞神經併發症,造成至少55名死亡病例。腸病毒71型主要經由口糞傳染,在兒童及嬰兒造成的臨床症狀包括口手足症、無菌性腦膜炎(aseptic meningitis)、腦炎(encephalitis)、類小兒麻痺脊髓炎(polio-like myelitis)。由於尚無一個合適的動物模式,以致目前腸病毒71型的感染造成中樞神經的致病機轉仍不清楚。為了瞭解腸病毒的散佈途徑及致病機轉,我們建立了一個經由口服感染腸病毒71型的小鼠動物模式。我們首先證實腸病毒71型能夠感染腸道細胞株Caco-2,在以臨床分離株4643、Caco-2適應株、及小鼠適應株分別感染體外培養的新生鼠小腸道,發現可以在感染2天後偵測到病毒VP1抗原;其中以小鼠適應株最強,Caco-2適應株次之。由於適應株病毒能夠成功感染新生鼠腸道,我們將小鼠適應株以口服餵食1 x 106 PFU 至一天大的新生鼠,在感染後6至9天,50 %餵食小鼠適應株的新生鼠產生後肢麻痺的症狀並死亡,但餵食原來4643株的組別除了皮膚發生病灶,並無其他併發症產生。如果餵食病毒量增加至1 x 107 PFU,則在感染後第3天可以達到100 %的死亡率。我們也可以在感染小鼠的小腸、血液、脾臟、腦、腦幹、脊髓、肺、心臟、皮膚、及後肢肌肉中分離出腸病毒71型。後肢麻痺的病鼠在小腸、腦幹、脊髓等區域都發現單核球細胞浸潤。動態的分析顯示,感染後會有二次的病毒血症,分別出現在感染後6小時及24小時;小腸外肌肉層最先出現VP1抗原,之後才出現在胸椎及腰椎,最後出現在腦幹。另外我們也發現腸病毒71型在同一胎新生鼠中可以透過口糞,或有親密的接觸而傳染。此種新生鼠感染模式非常類似臨床腸病毒71型的感染,它不僅可幫助我們瞭解腸病毒的致病機轉,也可作為抗腸病毒71型藥物的篩檢。
英文摘要 Enterovirus 71 (EV71), a single positive strand RNA virus, belongs to Picornaviridae. Since the first epidemic in 1973, there have been several outbreaks of hand-foot-mouth disease (HFMD) caused by EV71 in the United States, Australia, and East Asia. An outbreak at Taiwan in 1998, caused 55 children die with central nervous system (CNS) involvement. The fecal-oral route is thought to be the predominant mode of enterovirus transmission. EV71 has been associated with an array of clinical syndromes including HFMD, aseptic meningitis, encephalitis, polio-like myelitis, and paralysis among infants and children. Its pathogenesis is not clearly understood. There is also no animal model to understand how enterovirus spreads to the central nervous system (CNS). Therefore, we established a murine model of EV71 infection by oral route to understand viral spread in vivo. EV71 can infect intestinal epithelial cell line Caco-2 and neuroblastoma cell line SK-N-SH. The cell- or mouse-adapted EV71 was used to infect murine neonatal intestine ex vivo. The EV71 viral protein 1 (VP1) was detected in intestine epithelial cells 2 days post infection, and stronger staining was observed in tissues infected with mouse-adapted strain than those infected with Caco-2 adapted or parental one. Since mouse-adapted EV71 can successfully infect murine neonatal intestine, we further explored the EV71 infection orally. Oral inoculation of 1 x 106 PFU mouse-adapted EV71 into neonate mice caused flaccid paralysis and death in 50 % of mice at 6-9 days post infection while parental EV71 only induced mild skin lesions. The mortality increased to 100 % at day 3 post infection when mice were inoculated with 1 x 107 PFU of virus. EV71 can be re-isolated from intestine, blood, spleen, brain stem, brain, spinal cord, heart, liver, lung, skin, and hind limb muscle of the infected mice. The pathological analysis found cell infiltration in brain stem, spinal cord and intestine. The kinetic study showed that two-phase viremia appeared at 6 and 24 hours post infection, respectively. The EV71 VP1 antigen first occurred in intestinal muscularis externa, then in cervical and lumbar spinal cord, and finally in the brain stem. Furthermore, EV71 can spread between littermates by fecal route or close contact. This animal model mimics the clinical EV71 infection, and can be used for further study of EV71 pathogenesis and screening of antiviral drugs.
論文目次 目錄
中文摘要 i
英文摘要 iii
致謝 v
目錄 vi
表目錄 viii
圖目錄 viiii

第一章 緒論 1

第二章 材料與方法 8
 A 細胞株 8
  A-1 細胞株 8
  A-2 細胞培養 8
 B、病毒株 10
  B-1 病毒來源 10
  B-2 病毒的感染 10
  B-3 病毒的適應(adaptation) 11
  B-4 腸病毒71型效價測定(Titration of enterovirus 71) 11
 C、實驗動物 12
 D、體外器官培養感染模式 (Intestinal organ culture; IOC) 12
 E、以腸病毒71型感染ICR之新生鼠(Infection of neonatal ICR mice by enterovirus 71) 13
 F、組織切片的製備 (preparation of tissue sections) 13
  F-1 石蠟切片(paraffin sections) 13
  F-2 冷凍切片(cryosections) 14
 G、免疫組織化學染色(Immunohistochemistry stain) 14

第三章 結果 16
 A. 腸病毒71型在不同細胞中的生長速率 16
  1. 6種臨床病毒株在Vero、Caco-2及SK-N-SH細胞株中複製的情形 16
 B. 腸病毒71型感染體外培養之新生小鼠腸道 17
  1. 腸病毒71型體外感染新生鼠腸道 17
 C. 腸病毒71型感染新生小鼠 18
  1.病徵 18
  2.腸病毒在各器官的分佈 19
  3. 小鼠適應型病毒株4643在感染新生鼠中的動態散佈 20
 D. 新生小鼠間腸病毒71型的傳播 20

第四章 討論 22
第五章 參考文獻 29

表 36
圖 39
附錄1 56
附錄2 57


表目錄
表1. The clinical presentation of six EV71 isolates used in this study 36
表2. Three cell lines used in this study 37
表3. Summary of EV71 VP1 positive stain in different tissues of infected mice 38


圖目錄
圖1. EV71 clinical isolates can replicate in vero, SK-N-SH, and Caco-2 cell lines 39
圖2. Micrographs of EV71 infection of neonatal murine small intestine ex vivo 40
圖3. The appearance of EV71-infected mice by oral route 41
圖4. Organ distribution of EV71 VP-1 antigen in orally infected mice 42
圖5. Mortality of mice after EV71 infection 43
圖6. Pathologic changes in EV71 infected mice 44
圖7. Tongue protruding in EV71 infected mice 45
圖8. Re-isolation of EV71 from various organs of EV71-infected mice 46
圖9. EV71 viremia kinetics 47
圖10. EV71 VP1 staining in jejunum 48
圖11. EV71 VP1 staining in rectum 49
圖12. EV71 VP1 staining in brain stem 50
圖13. EV71 VP1 staining in cervical spinal cord 51
圖14. EV71 VP1 staining in thorax spinal cord 52
圖15. EV71 VP1 staining in lumbar spinal cord 53
圖16. EV71 VP1 staining in skin 54
圖17. Transmission between littermates of EV71-infected pup. 55
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