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系統識別號 U0026-0812200910433738
論文名稱(中文) 腸病毒71型之突變株對小鼠毒性之影響
論文名稱(英文) The toxic effect of an Enterovirus 71 mutant in mice
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 91
學期 2
出版年 92
研究生(中文) 周春婷
研究生(英文) Chun-Ting Chou
電子信箱 elisa78.tw@yahoo.com.tw
學號 s4690110
學位類別 碩士
語文別 中文
論文頁數 76頁
口試委員 指導教授-余俊強
口試委員-蘇益仁
口試委員-黎煥耀
中文關鍵字 腸病毒71型  動物模式  致病機轉 
英文關鍵字 animal model  enterovirus 71  pathogenesis 
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 型感染小孩主要可造成手口足症(hand-foot-mouth disease; HFMD) ,嚴重時伴隨腦炎
(encephalitis)、無菌性腦膜炎(aseptic meningitis)、肺水腫(pulmonary edema)及類小兒麻痺性的脊髓炎及麻痺。本實驗室已成功建立7 天大的ICR 小鼠以口服感染病毒71 型的動物模式。在感染後4~6 天可呈後肢麻痺等神經症狀,約在感染後7~8 天死亡,死亡率約50~80%。在此動物模式中所採用的腸病毒71 型為一小鼠適應株(EV71/MP4),其原始株(EV71/4643)並不能以口服的方式感染7 天大的ICR 小鼠,此兩種病毒皆能以腹腔注射的方式感染1 天大的小鼠,造成小鼠死亡,分析這兩種病毒的差異發現EV71/4643 的半致死劑量(LD50)為104,而EV71/MP4為102,兩者相差100 倍,其所造成的病毒斑也發現EV71/MP4 大於EV71/4643,觀察這兩種病毒在human neuroblastoma (SK-N-SH)、human
colorectal adenocarcinoma (Caco-2)、human rhabdomyosarcoma(RD)細胞株的生長曲線可發EV71/MP4 的生長速度高於EV71/4643。經由MP4 與4643 的定序結果發現不論是核 酸或胺基酸兩者的相似度均高達99%,以其他腸病毒71 型之病毒株進而分析5`端的非轉譯區(5`-unntranslation region)發現有6 個突變位,而其中4643 及MP4在internal ribosome entry site (IRES)上具有3 個突變位分別是nt:307(C toT)、nt: 498 (A to G)及nt: 577(C to T) 。我們發現在感染SK-N-SH 細
胞後12 小時,MP4 比4643 更具毒力且更能誘發SK-N-SH 細胞產生細胞凋亡的現象。而4643 及MP4 要48 小時感染後,才能誘發Caco-2 細胞凋亡的現象。進一步以4643 及MP4 以餵食方式感染7 天大的ICR 小鼠,我們發現感染4643 的小鼠無法分離出病毒,但感染MP4 的小鼠在第3、7 天在腦、脊髓和骨骼肌分離到病毒。顯示4643 無法以餵食方式感染7 天大的ICR 小鼠。由以上結果我們推論IRES 在MP4 的毒力上扮演很重要的角色。
RNA interference(RNAi)經由雙股RNA 的作用,對序列具有高度的專一性,在動物及植物細胞的研究中發現可分解特定的RNA。我們設計了6 個siRNA(small interfering RNA),發現siRNA-2、siRNA-3、siRNA-2+3的SK-N-SH 細胞,在感染腸病毒71 型後24 小時其細胞病變效應
(cytopathic effect) 不明顯及且細胞存活率增加。這樣的結果顯示RNAi可提供對抗對腸病毒71 型一個有效的策略。
英文摘要 Enterovirus 71(EV71), a single positive strand RNA virus, belongs to Picornaviridae. EV71 infection manifests most frequently as the childhood exanthem known as hand-foot-and-mouth disease (HFMD), as well as a number of severe diseases, including encephalitis, aseptic meningitis, pulmonary edema and poliomyelitis paralysis. We have demonstrated that a
mouse-adapted enterovirus 71 strain, EV71/4643/MP4 (MP4), but not the parental strain EV71/4643 (4643), could orally infect and induce
neurological diseases in 7-day-old ICR mice with a mortality rate of 50~80%. Therefore, the purpose of the present study was to define the
virulence of EV71/MP4 strain. MP4 was found to be more virulent than 4643 in 1-day-old mice with an increased mortality and decreased survival
time (LD50: 102 pfu/mouse and 104 pfu/mouse, respectively). MP4 had a more rapid growth rate and exhibited a larger plaque size than 4643 in
SK-N-SH (human neuroblastoma), CaCo-2 (human colorectal
adenocarcinoma), and RD (human rhabdomyosarcoma) cells. Comparison of nucleotide and amino acid sequence revealed 99% identity. Nucleotide (nt)
sequence analysis revealed that there were six point mutations on the 5`-untranslated region (UTR) of the other EV71. Comparison of nucleotide
sequence of internal ribosome entry site (IRES) with 4643 and MP4, there were three point mutation: nt:307(C to T), nt:498 (A to G), and nt:577(C to T). We found both 4643 and MP4 induced SK-N-SH cells undergo apoptosis at early stage, but induced Caco-2 cells apoptosis at late stage. Our results also indicated MP4 was more virulent than 4643 at 12 hour postinfection.
To further compare in vivo virulence of EV71/4643 and EV71/MP4, We orally infected 7-day-old ICR mice with 4643 or MP4. There was no virus
detected in 4643-infected mice. However, virus could be isolated from the brain, spinal cord and skeletal muscle of MP4-infectd mice at 3 and 7 day
postinfection. These results indicted that MP4, but not 4643 could orally infect 7-day-old ICR mice. Because our results indicated MP4 has more
replication rate and neurovirulence than 4643 in vitro and in vivo, we assume that the mutation sites on the IRES of MP4 may play an important role in viral virulence.
RAN interference (RNAi) is the process by which double-stranded RNA directs sequence-specific degradation of messenger RNA in animal and plant cells. We have designed six EV71-specific siRNA-1 to –6, and demonstrated that siRNA-2、siRNA-3 and the combination of siRNA-2 and -3 could effectively protect SK-N-SH cells against 4643 infection. Pre-treatment of SK-N-SH cells with the siRNAs markedly reduced the EV71-induced cytopathic effect and increased cell viability at 24 hours after infection. Thus, the specific intracellular antiviral resistant elicited by siRNA may provide a therapeutic strategy against evterovirus 71.
論文目次 中文摘要--------------------I
英文摘要--------------------III
目錄------------------------V
表目錄----------------------VII
圖目錄----------------------VIII
第一章緒論------------------1
第二章研究動機與實驗設計----9
第三章材料與方法------------11
A 材料----------------------11
A-1 實驗動物----------------11
A-2 病毒--------------------11
A-3 細胞株------------------11
A-4 培養液及相關藥品--------11
A-5 其他試劑----------------14
A-6 耗材--------------------15
A-7 儀器--------------------15
B 方法----------------------17
B-1 細胞培養----------------17
B-2 病毒培養----------------19
B-3 組織切片之備製----------21
B-4 細胞凋亡之分析----------22
B-5 蛋白質的分析------------24
B-6 逆轉錄聚合連鎖反應------27
B-7 5` RACE system ---------31
B-8 細胞株之DNA轉殖---------34
第四章結果------------------35
A. 4643 及MP4 其nucleotide 及amino acid 的序列相似度為99% --------------------35
B. 腸病毒71 型感染SK-N-SH 細胞造成細胞死亡------------------------------------35
C. 腸病毒71 型感染SK-N-SH 及Caco-2 細胞可誘發細胞凋亡------------------------36
C-1 以Annexin V 偵測MP4 所引起的細胞凋亡之現象--------------------------------37
C-3 感染SK-N-SH 細胞以西方墨點法偵測Caspase 9 的活化--------------------------38
C-4 感染Caco-2 細胞以西方墨點法偵測Caspase 9 的活化--------------------------38
D. 4643 無法以經口感染的方式感染7 天大的ICR 小鼠------------------------------39
E. 利用核酸干擾現象(RNA interference, RNAi)可抑制腸病毒71型在SK-N-SH 細胞的感染------------------------------------------------39
第五章討論------------------42
第六章參考文顯--------------48
表--------------------------55
圖--------------------------57
附錄一----------------------74
附錄二----------------------75
附錄三----------------------76

表目錄
表一. Comparison of sequence identity of 4643 with MP4 -------------------55
表二. The mutation sites of 5`UTR of the EV71 isolates -------------------56
圖目錄
圖一. Alignment of 5`-UTR from various EV71 isolates -------------------57
圖二. Viability of 4643 or MP4 infected SK-N-SH cells ----------------------58
圖三. MP4 induces SK-N-SH cells apoptosis in a dose dependent manner-------59
圖四. EV71 induces apoptosis of Caco-2 and SK-N-SH cells--------------------60
圖五. Detection of EV71 replication in the SK-N-SH and Caco-2 cells --------61
圖六. EV71/4643 and EV71/MP4 induce apoptosis of SK-N-SH and Caco2 cells-----62
圖七. EV71 induce Caspase 9 activation in SK-N-SH and Caco-2 cells.-----------63
圖八. EV/MP4 induced neurological symptoms on ICR mice------------------------64
圖九. Re-isolation of EV71 from various organs of EV71-infected mice----------65
圖十. RNAi inhibits EV71 infection in SK-N-SHcells --------------------66
圖十一. RNAi inhibits EV71 infection in SK-N-SH cells ----------------------67
圖十二. RNAi inhibits EV71 infection in SK-N-SH cells ----------------------68
圖十三. RNAi inhibits EV71 infection in SK-N-SH cells ----------------------69
圖十四. RNAi inhibits EV71 infection in SK-N-SH cells ----------------------70
圖十五. RNAi inhibits EV71 infection in stable cells ----------------------71
圖十六. RNAi inhibits EV71 infection in stable cells ----------------------72
圖十七. RNAi inhibits EV71 infection in stable cells ----------------------73
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