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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張淑媛 | |
| dc.contributor.author | Ting-Wei Chang | en |
| dc.contributor.author | 張庭瑋 | zh_TW |
| dc.date.accessioned | 2021-06-15T12:27:15Z | - |
| dc.date.available | 2021-08-26 | |
| dc.date.copyright | 2016-08-26 | |
| dc.date.issued | 2016 | |
| dc.date.submitted | 2016-08-09 | |
| dc.identifier.citation | 1. S. J. Flint , V.R.Racaniello, L. W. Enquist , A. M. Skalka, Principles of virology. human immunodeficiency virus pathogenesis. Vol. 2. 2009.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49985 | - |
| dc.description.abstract | HIV-1高效率抗反轉錄病毒治療(highly active antiretroviral therapy, HAART)能有效降低病人的致死率與發病率。然而因有些病人服藥順從性不佳及病毒本身高突變率的因素，使抗藥性病毒株的盛行率也增加。在台灣，HARRT第一線用藥主要是兩種NRTI(nucleoside reverse transcriptase inhibitor)藥物加一種NNRTI(non-nucleoside reverse transcriptase inhibitor)藥物。因為NNRTI的基因屏障比較低，且目前已知主要的NNRTI藥物抗藥性突變會造成多種NNRTI藥物感受性下降，所以NNRTI藥物抗藥性的發生率也逐年增加。除了主要的抗藥性位點之外，相關的突變也隨之被觀察到，但是這些相關突變對病毒生長與藥物感受性之影響仍不清楚。
本實驗室首先針對2008-2015台灣地區5114條HIV-1反轉錄酶序列進行分析。NNRTI主要突變以反轉錄酶上K103、V179、V106與L100為多數。本實驗目的為確認L100I/V是否確實對NNRTI有抗性，並想進一步了解多型性突變I202V之影響。首先我們比較病毒生長能力，發現HXB2/L100V與HXB2/L100V+I202V病毒複製能力明顯低於原生型HXB2病毒；同時發現HXB2/L100V與HXB2/L100V+I202V反轉錄酶活性也顯著低於帶有其它突變位點之病毒反轉錄酶活性。HXB2/L100V與HXB2/L100I對於NNRTI藥物 NVP、EFV與ETR皆具有抗性。HXB2/L100V/I加入I202V多型性突變後，會增加對NVP與ETR的感受性；相反的HXB2/L100V/I+I202V卻會降低對EFV的感受性。 總結本研究結果，反轉錄酶上L100V與L100I點突變，的確會降低反轉錄酶活性，進而影響病毒複製的能力，也會造成病毒對於NNRTI的感受性下降。 | zh_TW |
| dc.description.abstract | The morbidity and mortality of HIV-1-infected indviduals has reduced after introduction of highly active antiretroviral therapy (HAART). However, the poor adherence of HIV patients and the high mutation rate of HIV-1 result in the increased prevalence of drug-resistant viruses. In Taiwan, the first line regimen of HAART consists of 2 nucleoside reverse transcriptase inhibitors (NRTIs), and one non-nucleoside reverse transcriptase inhibitor (NNRTI). Due to the lower genetic barrier of NNRTI and the major NNRTI mutations often caused cross resistance to other drugs in the same category, the prevalence of NNRTIs resistant virus has increased recently. Nevertheless, the impacts of some NNRTI related mutations and polymorphisms on drug susceptibility and virus fitness are still unclear.
In this study, a total of 5114 HIV-1 reverse transcriptase sequences amplified from HIV-1 infected, treatment-naive patients from 2008 to 2015 were analyzed. Among these NNRTI-related major mutations, K103, V179, V106 and L100 were dominant.We aimed to determine the effects of L100V/I mutation and its compensatory mutations on virus fitness and susceptibility to NNRTIs. First, the HXB2/L100V and HXB2/L100V+I202V viruses showed slight impairments in replication as well as reverse transcriptase activity when compared to that of the parental wild-type HXB2 viruses. In virus susceptibility to NNRTIs, HXB2/L100V and HXB2/L100I had reduced susceptibility to NVP, EFV and ETR. These virus harbored I202V mutations also showed increased susceptibility to NVP and ETR. Neverless, HXB2/L100V+I202V and HXB2/L100I+I202V reduced virus susceptibility to EFV when compared to that of the single L100-mutant viruses. In addition, HXB2 / L100V and HXB2 / L100I mutations had no effect on viral susceptibility to RPV. In summary, the presence of L100V/I mutationreduced reverse transcriptase activity and virus replication capacity. The virus susceptibility to NNRTIs was also decreased. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T12:27:15Z (GMT). No. of bitstreams: 1 ntu-105-R03424022-1.pdf: 2371052 bytes, checksum: cb6022c000d28f166f6a84e2e0ea73a1 (MD5) Previous issue date: 2016 | en |
| dc.description.tableofcontents | 致謝 II
中文摘要 III 英文摘要 IV 目錄 VI 圖目錄 IX 表目錄 X 附錄 XI 第一章 緒論 1 1-1 人類免疫缺陷病毒第一型(HIV-1)與後天免疫缺乏症候群(AIDS) 1 1-1-1 病毒簡介 1 1-1-2 病毒傳染途徑與致病機制 1 1-2 HIV-1 流行現況 1 1-3 高效率抗反轉錄病毒治療(highly active antiretroviral therapy , HAART) 2 1-3-1 高效率抗反轉錄病毒治療簡介 2 1-3-2 反轉錄酶抑制劑(reverse transcriptase inhibitor, RTI) 2 1-3-3 蛋白酶抑制劑 (protease inhibitor, PI) 5 1-3-4 嵌入酶抑制劑 (integrase inhibitor, InSTI) 5 1-3-5 其他抗病毒藥物 6 1-3-6 現行藥物組合 6 1-4 HIV-1抗藥性概況 7 1-4-1 抗藥性突變產生 7 1-4-2 反轉錄酶抑制劑抗藥性突變 7 1-5 研究動機 9 第二章 實驗材料與方法 10 2-1 實驗材料 10 2-1-1 細胞 10 2-1-2 商業試劑 10 2-1-3 抗體 11 2-2 實驗方法 11 2-2-1 病毒RNA萃取 (viral RNA extraction) 11 2-2-2 RNA反轉錄反應 (reverse transcription) 12 2-2-3 HIV-1 B亞型病毒Gag-Pol基因巢式聚合酶連鎖反應 (Nested-PCR) 12 2-2-4 pCRTMII-TOPO®連接(Ligation)反應 13 2-2-5 定位突變實驗 (Site-directed mutagenesis) 13 2-2-6 轉型作用(Transformation) 13 2-2-7 小量細菌質體萃取(Mini plasmid extraction) 13 2-2-8 大量細菌質體萃取(Maxi plasmid extraction) 14 2-2-9 洋菜膠萃取DNA片段( DNA gel purification) 14 2-2-10 T4 DNA連接酶反應(T4 DNA ligase ligation) 15 2-2-11 293T細胞株培養 15 2-2-12 轉染作用(Transfection) 15 2-2-13 蛋白質電泳(SDS-polyacrylamide gel electrophoresis , SDS-PAGE) 16 2-2-14 西方墨點法(western blot) 16 2-2-15 周邊血液單核細胞分離與培養 (PBMC purification) 17 2-2-16 p24酵素結合免疫吸附分析(p24 enzyme-linked immunosorbent assay, ELISA) 17 2-2-17 周邊血液單核細胞感染實驗(PBMC infection) 18 2-2-18 U87-CD4+-CXCR4+細胞株培養 18 2-2-19 U87-CD4+-CXCR4+細胞株感染實驗(U87-CD4+-CXCR4+infection) 19 2-2-20 單步驟即時定量聚合酶連鎖反應(One-Step real-time PCR) 19 2-2-21 製備結晶紫(crystal violet)洋菜膠與電泳 19 2-2-22 反轉錄酶分析(Reverse Transcriptase Assay) 20 2-2-23 統計方法與分析軟體 20 第三章 實驗結果 21 3-1 2008-2015台灣非核苷酸反轉錄酶抑制劑抗藥性突變位點統計分析 21 3-2 2008-2015年台灣HIV-1反轉錄酶L100V/L100I族群數量分析 22 3-3 2011-2015年台灣HIV-1 B亞型多型性變異統計分析 22 3-4 建構HIV-1 pHXB2定位突變質體 23 3-5 HIV-1Gag蛋白質表現未受到反轉錄酶點突變影響 23 3-6 抗藥性反轉錄酶點突變病毒於U87-CD4+-CXCR4+之生長能力 24 3-7 抗藥性反轉錄酶點突變病毒於人類周邊血單核細胞之生長能力 24 3-7-1 測試合適健康人類周邊血單核細胞捐贈者 24 3-7-2 人類周邊血單核細胞(PBMC)中抗藥性反轉錄酶點突變病毒生長能力 25 3-8 反轉錄酶點突變對反轉錄酶活性影響 25 3-9 反轉錄酶點突變對NNRTI藥物感受性影響 25 3-9-1 PBMC中反轉錄酶點突變對藥物感受性影響 26 3-9-2 U87-CD4+-CXCR4+細胞株中反轉錄酶點突變對藥物感受性影響 27 3-10 不同突變株的反轉錄酶對於非核苷類似物反轉錄酶抑制劑抑制能力影響 29 第四章 實驗討論 30 第五章 參考文獻 36 | |
| dc.language.iso | zh-TW | |
| dc.title | 探討人類免疫缺乏病毒非核苷酸類似物抑制劑抗藥性突變L100V/L100I與多型性突變之功能性影響 | zh_TW |
| dc.title | Functional characterization of HIV-1 NNRTI-resistance
mutation L100V/L100I and related polymorphic mutations | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 104-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 高全良,李君男,劉旻禕,何炳慶 | |
| dc.subject.keyword | 非核?酸類似物抑制劑,L100V,L100I, | zh_TW |
| dc.subject.keyword | NNRTI,L100V,L100I, | en |
| dc.relation.page | 71 | |
| dc.identifier.doi | 10.6342/NTU201601261 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2016-08-09 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
| 顯示於系所單位： | 醫學檢驗暨生物技術學系 | |
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