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標題: | 發展微小干擾核醣核酸來抑制A型流行性感冒病毒繁殖 Developing Small Interfering RNA to Intervene Influenza Virus Type A Replication |
作者: | Ping-Sheng Wu 吳秉昇 |
指導教授: | 黃立民(Li-Min Huang) |
共同指導教授: | 張鑾英(Luan-Yin Chang) |
關鍵字: | 流行性感冒病毒,微小干擾核醣核酸, influenza virus,small interfering RNA, |
出版年 : | 2007 |
學位: | 碩士 |
摘要: | 研究背景:A型流行性感冒病毒估計每年可感染全球人口的20 %,甚至可能因基因重組造成全球大流行。疫苗是用來預防A型流感主要方式,然而疫苗株台灣地區卻僅有53-82 %的保護率,且由於A型流感病毒具有高突變率,使得抗病毒藥物及疫苗很容易失效,因此發展新的預防或治療方式是目前當務之急。微小干擾核醣核酸是細胞用來調控核醣核酸的機制,使得一些核醣核酸不能夠執行其功能。而A型流感病毒之基因片段中有一些長久保存不變的序列已執行重要的功能,這將是微小干擾核醣核酸理想的抑制目標。
研究方法:在兩個A型流感病毒株(A/Panama/1/68, H3N2; A/WSN/33, H1N1)的目標基因(NS1 gene,PA gene,NP gene)之片段中選擇出數個較具高度保留的區域,並在此之間找出適合作為siRNAs標的序列的二十八個核酸序列,重組為可表現short hairpin RNA(shRNA)之質體,並使用轉染溶液jetPEI、Lipofectamine 2000及lentivirus進行MDCK-ATCC與MDCK-p69細胞株轉染。經由紅血球凝集測試、定量PCR或病毒斑分析等方式,觀察其是否具有抑制流感病毒的效果。若找出數個較佳抑制病毒效果的微小干擾核酸之序列,亦進行細胞之共同轉染以觀察有無加成作用。 結果:利用流式細胞儀可發現使用jetPEI與Lipofectamine 2000對MDCK-ATCC之轉染效率各約為20-30 %及34%,轉染效率與繼代數呈反比。文獻中發現具抑制效果之NP-1496 siRNA,需大量經jetPEI轉染才可發現病毒抑制效果 (50-75% HA unit decrease)。H3N2 (A/Panama/1/68) siRNA之研究中十四段標的序列之轉染可見PA-1’序列最具有病毒抑制效果 (50% HA unit decrease),PA-2、NP-3與PA-4’序列則亦於紅血球凝集測試中具病毒抑制效果,而較有效之shRNA質體共同轉染之組合則並無加成作用。PA-1’於病毒斑分析中亦可見抑制效果 (67% plaques decrease)。H1N1 (A/WSN/33) siRNA之研究中於MDCK-p69細胞株轉染,可見PA-1’與NP-1496序列於紅血球凝集測試中有部份病毒抑制效果 (50-75% HA unit decrease)。若以pseudotyped lentivirus與puromycin進行MDCK-p69細胞之感染與篩檢,則可將轉染效率提高至74%,而序列PA-1’與NP-3皆可見到較明顯之病毒抑制效果 (50-88% HA unit decrease)。shRNA序列之抑制效果與否與標的序列之3’端為stem或loop 結構較無明顯關聯。 結論: PA-1’為較有效抑制A/Panama/1/68, H3N2或A/WSN/33, H1N1之標的序列,其餘序列則出現效果分歧的結論,而更換環狀序列並無增進抑制效果之成效。shRNA expression cassette抑制病毒效果與質體轉染效率、細胞繼代數及轉染後感染之時間點有關,而lentivirus系統轉染之效率優於liposome系統。即使是鄰近序列,對於病毒抑制的效果也可能有很大的差別。標的基因3’端之二級結構與病毒抑制效果之間的關係仍需要更多序列分析。 Background. Type A influenza virus infects 20% of global population every year, and genetic reassortment between different strains could cause pandemic outbreak. Vaccine is the only available strategy for type A influenza prevention, however, the identity between vaccine and circulating strain is only 52 to 82% in Taiwan. Anti-influenza drugs are not reliable due to highly mutant character of type A influenza virus. Therefore, a new strategy for prophylaxis and treatment for type A influenza virus should be studied. Small interfering RNAs (siRNAs), which target the more conserved sequence of type A influenza virus, could knockdown the gene expression and virus replication. Plasmid or lentivirus vectors carriage with short hairpin RNA cassette might more persistently express the shRNA and inhibit the virus. Materials and Methods. The target genes (NS1 gene, PA gene and NP gene) of two type A influenza virus strains, A/WSN/33 (H1N1) and A/Panama/1/68(H3N2), are aligned for the siRNA target sequences. MDCK-ATCC and p69 cell lines are then transfected with quantitative shRNA plasmid, followed by influenza A virus infection. Pseudotyped lentivirus with shRNA sequence is used as an alternative tool for cell transduction. The viral load of supernatant are analyzed by hemagglutiation assay (HA assay), real-time PCR and plaque assay. If several potently inhibitory sequences could be found, the inhibitory effect of co-transfection is also studied. Results. For MDCK-ATCC cell, the transfection efficiencies of jetPEI and Lipofectamine 2000 are 20-30% and 34%, respectively, which were inverse to the passage number. Large amount NP-1496 siRNA (500pmol) via jetPEI delivery is related to influenza virus inhibition (50-75% HA unit decrease). In H3N2 (A/Panama/1/68) shRNA study, PA-1’ is the most powerful sequence for influenza virus inhibition, which reveals 50% and 67% influenza virus decrease, respectively. PA-2,NP-3 and PA-4’ also have some inhibitory effects. The co-transfection reveal no synergistic effect. In H1N1 (A/WSN/33) shRNA study, PA-1’ and NP-1496 sequences could achieve 50-75% HA unit decrease after transfection to MDCK-p69 cell. The transfection efficiency could increase to 74% after pseudotyped lentivrus transduction and puromycin selection. PA-1’ and NP-3 sequences reveal more obvious influenza virus inhibition (50-88% HA unit and 57-91% plaques decrease). There is no relationship between the virus inhibition and secondary structure of 3’ end of target sequences in this study. Conclusion. PA-1’ sequence is a more effective sequence in type A influenza virus inhibition. However, some sequences reveal diverse conclusion. No more effect could be achieved after changing the loop sequence of shRNA. The knockdown effect of shRNA plasmid is related to the transfection efficiency, cell passage number and timing of virus infection. The transfection efficiency is better in lentivirus system than in liposome system. The inhibition effect could be completely different between two nearby sequences. The relationship between secondary structure of 3’ end of target sequence and virus inhibition should be further surveyed. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/27958 |
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顯示於系所單位: | 臨床醫學研究所 |
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