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研究生：	吳孟采 Wu, Meng-Tsai
論文名稱：	利用小片段干擾核苷酸引發之核苷酸干擾現象抑制腸病毒71型的感染 Inhibition of enterovirus 71 infection by small interfering RNA-mediated RNA interference
指導教授：	張文粲 Chang, Wen-Tsan
學位類別：	碩士 Master
系所名稱：	醫學院 - 生物化學研究所 Department of Biochemistry
論文出版年：	2003
畢業學年度：	91
語文別：	中文
論文頁數：	86
中文關鍵詞：	小干擾型RNA 、第三型RNA聚合酶、RNA干擾現象、腸病毒71型
外文關鍵詞：	enterovirus 71, RNA interference, RNA polymerase III, small interfering RNAs
相關次數：	點閱：141 下載：1
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小干擾型RNA(small interfering RNAs, siRNAs)所引發的RNA干擾現象是一種強而有力將目標基因沉默的方法。相關文獻指出，在哺乳動物細胞中以H1與U6 promoter所轉錄的siRNAs可有效抑制基因表現，且不會引起抗病毒反應。我們分別建構人類U6、H1 promoters與老鼠U6、H1 promoters用來驅動正義與反義小單股RNA、小雙股RNA與小髮夾型RNA四種不同結構的小RNA分子，以螢光酵素基因為標定序列，藉由量化的數值來觀察與比較四種promoters轉錄出來的四種小分子RNA:小髮夾型RNA與小雙股型RNA﹔正義與反義單股RNA，抑制基因表現上的差異。由結果知道，正義與反義小單股RNA無法有效抑制基因表現，小雙股RNA與小髮夾型RNA可以抑制基因表現，其中又以小髮夾型RNA抑制效果最明顯，然而，我們無法在luciferase 量化數值上看出四種promoter在調控轉錄上的差異。
腸病毒71型(Enterovirus 71, EV71)分類上屬小RNA病毒屬腸病毒種，在1969年加州腦炎嬰兒病例中首次被分離出來後，EV71被認為是造成，手、足、口病( hand、 foot、mouth disease-HFMD)，與一些地區性腦脊髓炎(encephalomyelitis)的主因，1998年台灣爆發大規模流行，超過90000個案例被報導出來，其中有55個病童死於腦中樞神經系統的病變。在腸病毒的治療上應首重於防止腸病毒的感染，本實驗室企圖以siRNA所引起的RNAi現象來抑制腸病毒71型的感染，首先，我們以Taiwan/4643腸病毒分離株為研究模型，由實驗結果可知si-EV71-2與si-EV71-3的siRNA表現質體可以抑制腸病毒分離株Taiwan/4643的感染，進一步將si-EV71-2與si-EV71-3siRNA表現框架連接一起，發現可以更有效抑制腸病毒感染。本實驗室已鑑別幾個不同分離株5 端不轉譯區，比較DNA序列上的差異，發現有6 個序列上的差異，然而這些差異無法有效的和不同分離株間毒性的差異作連接。

RNA interference is a sequence-specific posttranscriptional gene silencing mechanism. In mammalian cells, U6 and H1 promoters have been used to produce small interfering RNA. We have developed human and mouse RNA polymerase III H1, U6 promoters to drive shRNA, siRNA duplex, sense RNA and antisense RNA expression. Using pGL3-control as target vector, we examined the effect of firefly luciferase gene expression and compared the suppression efficiency by these small RNAs. We found that antisense RNA and sense RNA weakly inhibit gene expression, however siRNA duplex and shRNA efficiently suppress gene expression in SK-N-SH cell line. The transcriptional activities between these four Pol III promoters are not significantly different in SK-N-SH cell line.
Since its discovery in 1969, EV71 has been recognized as a frequent cause of epidemics of hand-foot-mouth disease (HFMD) associated with severel neurological sequelae in a small proportion of cases. In 1998, the largest EV71 outbreak in Taiwan, more than 90000 children with HFMD have been reported, and at least 55 died, suggesting neurovirulence of the pathogen. We focus on prevention of EV71 infection, and use small interfering RNA to inhibit EV71 infection. We found two siRNA target site could inhibit the Taiwan/4643 EV71 isolate infection. The inhibition of viral infection was even more efficient when two siRNA expression cassettes were linked together . Single nucleotide changes within the poliovirus IRES have been found to result in large alterations in neurovirulence. We compared the sequence of 5´NTR from three EV71 isolates, and found that there are six nucleotides different. However, these differences have no significant effect on the different neurovirulence between EV 71 isolates.

目錄
一. 序論:
1A.核苷酸干擾現象與沉默基因                  1
1B.以DNA 質體表現siRNA                       5
1C. RNAi 技術的應用與siRNAs載體的開發        7 
2A.腸病毒概論                                8
2B.腸病毒71 型流行病學與臨床症狀            10
2C.腦神經毒性                               11
二. 實驗源起
1.siRNA 表現質體與小分子RNA                 13
2.以siRNA 所引起的RNAi 現象抑制腸病毒感染   14
三. 材料與方法
A.材料                                      15
B.方法
一. 細胞的培養程序                          20
二. 基本分子生物技術                        21
三. 分析不同腸病毒分離株在5`NTR 序列上的差異26
四. 分析不同腸病毒分離株5`NTR 區域在調控轉譯
活性上的差異                                28
五. EV71siRNA 表現質體的構築                30
六. 以siRNA 表現質體抑制腸病毒感染          30
七. 建立持續表現siRNA 的穩定細胞(stable cell line)                                       31
八. 分離人類與老鼠的第三型RNA聚合酶驅動子                                          32
九. 比較不同長度的siRNA 序列在抑制基因表現上的差異                                          34
十. 不同驅動子(promoter) 在調控轉錄能力上的分析與比較                                        34
四.結果
一. 以PCR 方式將人類與老鼠U6 snRNA 、RNAse P H1
promoter 分離出來，構築成siRNA 表現質體                                          36
二. 以分離出來的人類與老鼠U6 snRNA 、RNAse P H1
promoters 轉錄的小分子RNA 抑制螢火蟲冷光基因的表現                                          36
三. 利用人類H1 promoter 轉錄26nt 與19nt siRNAs 抑制螢火蟲冷光基因表現，並比較抑制效果的差異  37
四. 比較人類與老鼠U6 snRNA 、RNAse P H1 promoter 所轉錄shRNA 結構在抑制基因表現上的差異      37
五. 不同siRNA 表現質體抑制Taiwan/4643 感染的情形                                          38
六. 經Taiwan/4643 感染後24 小時，以trypan blue 染色，計數細胞存活率                          39
七. 以Taiwan/4643 感染持續表現si-EV71-2 的穩定細胞(stable cell line)                          39
八. si-EV71-2-neo-6 與vector-1 細胞株Taiwan/4643 感染後24 小時，以trypan blue 染色，觀察細胞存活率                                          40
九. 不同腸病毒71 型分離株在5´NTR 序列的差異 40
十. 不同腸病毒71 型分離株在5´NTR 在調控轉錄活性上的差異                                      40
五. 討論                                    42
六. 參考文獻                                47
附圖                                        52
附表                                        75
附圖與附表
Fig.1 RNA 干擾現象與以後轉錄調控機制將基因沉默的作用模型                                      52
Fig.2 內生性小片段干擾核苷酸( Endogenous expression of siRNAs)                       53
Fig.3 U6 小核內RNA (U6 snRNA) 驅動子        54
Fig.4 RNA 水解酶P H1 ( RNAse P H1) 驅動子   55
Fig.5 構築siRNAs 表現質體                   56
Fig.6 以U6 promoters 轉錄的siRNAs 抑制luciferase 基因表現                                    57
Fig.7 H1 promoter 轉錄的siRNAs 抑制luciferase 基因表現情形                                    58
Fig.8 兩種luciferase 冷光基因siRNA 標定序列與對luciferase基因抑制的情形                    59
Fig.9 以不同promoters 轉錄的shRNA 抑制luciferase
基因表現的差異                              60
Fig.10 以Taiwan/4643 感染SK-N-SH 細胞       61
Fig.11 siRNAs 對腸病毒分離株-Taiwan/4643 感染SK-N-SH 細胞的影響                               62
Fig.12 siRNAs 對腸病毒分離株-Taiwan/4643 感染SK-N-SH 細胞的影響                               63
Fig.13 siRNAs 對腸病毒分離株-Taiwan/4643 感染SK-N-SH 細胞的影響                               64
Fig.14 siRNAs 對腸病毒分離株-Taiwan/4643 感染SK-N-SH 細胞的影響                               65
Fig.15 siRNAs 對腸病毒分離株-Taiwan/4643 感染SK-N-SH 細胞的影響                               66
Fig.16 siRNAs 對腸病毒分離株-Taiwan/4643 感染SK-N-SH 細胞的影響                               67
Fig. 17 SK-N-SH 細胞株經腸病毒分離株Taiwan/4643 感染24 小時後細胞存活率                       68
Fig.18 以腸病毒分離株-Taiwan/4643 感染vector-neo-1 與si-EV71-2-neo-6 細胞株                  69
Fig.19 以腸病毒分離株-Taiwan/4643 感染vector-neo-1 與si-EV71-2-neo-6 細胞株                  70
Fig.20 vector-neo-1 與si-EV71-2-neo-6 細胞株感染24 小時後細胞存活率                         71
Fig.21 腸病毒71 型原始分離株( BrCr ) 5 ’端不轉譯區的二級結構模型                            72  
Fig.22 腸病毒71 型分離株在5´端不轉錄區序列比對結果                                          73
Fig.23 不同腸病毒分離株5´端不轉錄區所調控轉譯的luciferase 活性比較                         74
Table.1 SK-N-SH 細胞經由不同處理的情形      75
                                    

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