臺灣博碩士論文加值系統

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小鼠以口服感染腸病毒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現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苷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 to T)、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。

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.

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 逆轉錄聚合酶連鎖反應 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. 利用核苷酸干擾現象(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-SH cells 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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