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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陶秘華(Mi-Hua Tao) | |
dc.contributor.author | Tzu-Hui Wu | en |
dc.contributor.author | 吳姿慧 | zh_TW |
dc.date.accessioned | 2021-06-12T18:36:01Z | - |
dc.date.available | 2009-08-08 | |
dc.date.copyright | 2007-08-08 | |
dc.date.issued | 2007 | |
dc.date.submitted | 2007-07-31 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/28055 | - |
dc.description.abstract | B型肝炎病毒(hepatitis B virus)為一種主要的人類致病病毒,全球有三億五千萬人為慢性感染者,有相當高的風險進程到肝臟衰退,肝硬化,及肝癌,但目前用來治療HBV慢性感染者的方法不盡理想,亟需發展新的治療方式。
RNA interference (RNAi),為長約19-23 nucleotides小片段的雙股RNA (siRNA)造成序列互補mRNA降解的現象。因為siRNA的有效性及序列特異性,RNAi不僅被當作一種工具用於基因功能及遺傳性疾病的研究,未來相當有潛力發展為抑制致病性病毒感染及複製的新型治療方式。 本實驗室先前已發現用1×1012vg/mouse 的劑量,以尾靜脈注射帶有shRNA的雙股腺相關病毒(dsAAV)可有效的抑制HBV基因轉殖鼠肝臟HBV蛋白、mRNA及DNA的表達至1%以下長達四個月,且未引起肝毒性及其他細胞激素的產生。根據Grimm et al.在2006年發表在Nature的一篇文章,發現利用dsAAV帶有U6 啟動子表達高劑量(1×1012vg/mouse ) shRNA時, shRNA會競爭肝臟內的內生性miRNA生產過程所需的成分exportin-5,而降低肝臟中miRNA的生成,影響細胞正常生理功能,進而造成肝臟損壞,在49株shRNA中有23株會引起老鼠死亡。本論文的研究希望在不引起毒性的情況下,提供慢性肝炎患者有效的 RNAi基因治療方式。以H1啟動子及U6啟動子表現有毒性的序列,發現在U6啟動子表現之下有毒性的shRNA,在H1 啟動子表現之下沒有毒性產生;另外,以H1啟動子表達時,在許多受測試的shRNA中, HBV-S1 shRNA具有最有效的HBV抑制能力 儘管HBV-S1 shRNA擁有有效的HBV抑制能力,有文獻指出在臨床病人的檢體中,在HBV-S1 shRNA治療的壓力下,對於HBV-S1有耐受性的突變型HBV可被篩選出來。為了更完全的抑制野生型及突變型HBV,採用同時表現多個shRNA的系統,表現抑制基因體不同區域或針對突變型HBV序列的shRNA。在我們所使用的兩種多個shRNA表達系統中,多啟動子表達系統有較好的抑制效果。在混合野生型及突變型HBV的情況下,同時表現針對野生型及突變型HBV的多shRNA表現系統抑制HBV的能力,與單獨表現野生型或突變型HBV的shRNA系統的抑制HBV能力不相上下。在此論文中,我們指出以H1啟動子表現shRNAi可能是較安全的基因治療方式,且野生型及突變型HBV的混合檢體可以被多shRNA表達系統抑制。 | zh_TW |
dc.description.abstract | Hepatits B virus (HBV) is a major human pathogen that chronically infected over 350 million people worldwide, rendering these patients at high risk of developing liver failure, cirrhosis and hepatocellular carcinoma. Current treatments for chronic HBV are suboptimal.
RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by means of sequence-specific mRNA degradation, triggered by small interfering RNA (siRNA) consisting of 19-23 nucleotide duplex. In cultured mammalian cells, RNAi has been proven effective in attenuating many human pathogenic viral infection and replication, including HBV. Because of its efficient and sequence-specific manner of gene silencing, RNAi has been considered as a potential powerful approach to treat viral infections in humans. In our previous study, a single administration of dsAAV2/8 vector, carrying HBV-specific shRNA, effectively suppressed the steady level of HBV protein, mRNA and replicative DNA in liver of HBV transgenic mice, leading to up to 2-3 log10 decrease in HBV load in the circulation and sustained for at least 120 days without any side effect. In contrast, a recent report using a similar dsAAV2/8 vector showed that high-level expression of shRNA caused saturation of exportin 5 and subsequent inhibition of endogenous pre-miRNA nuclear export, which proved to be lethal in 23 out of 49 mice. To examine the tocixity effect, we used both the H1 and U6 promoter to express the shRNA sequence in dsAAV2/8 vector. Our results showed that the toxicity effect can be eliminated by using the H1 promoter to replace the U6 promoter. Among several shRNAs tested, HBV-S1 shRNA possessed the most robust HBV suppression effect when driven by the H1 promoter. Despite the strong inhibition ability of HBVS1 shRNA, a recent report showed that shRNA resistant HBV mutant was selected out in the sample of a clinical patient during HBV-S1 shRNA treatment. In order to prevent the emergence of resistant viruses, we took the advantage of multiple shRNA expression system to express shRNA targeting against either the mutant HBV-S1 sequence or several separate locations on HBV genome. Between the two multiple siRNA expression systems we tested, the multiple promoter system exhibited better anti-HBV effect. More importantly when treating a mix populations of wild and mutant HBV, the multiple promoter system expressing two distinct shRNA showed better inhibition effect than that of the single shRNA expression system. In conclucsion, our results showed in dsAAV system shRNA driven by the H1 promoter could achieve an efficient antiviral effect without detectable toxicity, and the multiple shRN expression system was effective. | en |
dc.description.provenance | Made available in DSpace on 2021-06-12T18:36:01Z (GMT). No. of bitstreams: 1 ntu-96-R94424004-1.pdf: 4520374 bytes, checksum: e7ec82daad00a413552c9d9b8166b8bc (MD5) Previous issue date: 2007 | en |
dc.description.tableofcontents | 第一章、緒論
第一節、B型肝炎病毒簡介 1 1.1 B型肝炎病毒病毒學 1 1.2 B型肝炎致病機轉 3 1.3 慢性B型肝炎 3 1.4 現今用於慢性B型肝炎的治療方式 4 第二節、干擾RNA簡介 5 2.1 哺乳動物體內siRNA生成路徑 6 2.2 將siRNA送入細胞表達的方式 8 2.3 RNAi治療可能產生的副作用 9 第三節、已證實有明顯抑制HBV能力的siRNA序列及AAV系統 10 第四節、siRNA治療產生的突變型HBV 11 第五節、論文研究動機與目的 12 5.1 毒性研究 12 5.2 抑制HBV的有效性 13 第二章、材料與方法 14 第一節、質體的構築 14 1.1 質體的製備 14 1.2 1 % 洋菜膠電泳 14 1.3 DNA 片段分離 15 1.4 限制酶切割反應 15 1.5 填平5’端突出的Klenow反應 15 1.6 DNA接合反應 16 1.7 細菌熱休克轉殖法 16 第二節、細胞株培養與細胞表現檢驗 16 2.1 細胞株 16 2.2真核細胞的轉染 17 2.3細胞表現檢驗 17 第三節、包裝腺相關病毒 19 第四節、動物實驗 21 4.1實驗動物來源 21 4.2小鼠的注射方式 22 4.3小鼠的採血方式 22 第五節、動物檢體分析 23 5.1分離小鼠血清中HBV DNA 23 5.2小鼠血中HBV DNA的測定 23 5.3小鼠血中成份檢驗 23 5.4小鼠肝臟中的siRNA表達檢測 24 5.5小鼠肝臟中的AAV DNA檢測 24 5.6小鼠肝臟H.E.染色 25 第三章 實驗結果 27 毒性研究 27 第一節、以H1 啟動子表現shRNA的AAV系統 27 1.1 構築pAAV- H1/ HBV-S1-27、pAAV- H1/ sAg19、pAAV- H1/ sAg25 27 1.2測試將HBVS1 shRNA加長後的HBV-S1-27 shRNA的毒性 28 1. 3測試以H1啟動子表現sAg19 shRNA和sAg25 shRNA的毒性 29 第二節、測試以U6啟動子表現shRNA的AAV系統是否有毒性 30 2.1構築pAAV-U6/HBV-S1、pAAV-U6/GL2、pAAV-U6 /sAg19、pAAV-U6/sAg25 31 2.2測試U6啟動子表現sAg25 shRNA的毒性 32 第三節、測試不同siRNA序列在細胞內對於HBV的抑制能力 32 3.1細胞內以pSuper表現系統表現不同siRNA序列對於 HBV的抑制能力 33 3.2細胞內以pAAV系統帶有H1啟動子表現不同siRNA序列對於HBV的抑制能力 34 3.3細胞內以pAAV系統帶有U6啟動子表現不同siRNA序列對於HBV的抑制能力 34 3.4比較細胞內H1和U6啟動子的siRNA表達量 34 第四節、測試不同shRNA在HBV基因轉殖鼠中對於HBV的抑制能力 35 4.1 AAV帶有H1啟動子系統表現不同shRNA感染HBV基因轉殖鼠對於HBV抑制能力的比較 36 4.2比較老鼠體內H1啟動子的siRNA表達量和HBV抑制能力的關係 36 多個siRNA表現系統增加抑制HBV的有效性 37 第五節、以microRNA( miRNA)的系統表現多個siRNA 38 5.1檢驗表現單個siRNA的miRNA系統的抑制能力 38 5.2 GFP干擾HBV表現 39 5. 3測試pcDNA6.2/HBV-S1、pcDNA6.2/HBV-B4抑制HBV的能力 40 5.4比較以microRNA系統和shRNA系統表達siRNA的效果 41 第六節、以多個啟動子的表現多個siRNA 42 6.1以兩個H1啟動子表現HBV-S1及HBV-S2對於野生型及突變型HBV的抑制力 42 第四章 討論 44 4.1 毒性現象 45 4.2 siRNA表現量與毒性的關係 46 4.3 shRNA抑制HBV的有效性 47 4.4 shRNA抑制HBV的有效性與 siRNA的表達量的關係 48 4.5 對於同樣的目標基因miRNA系統抑制力不如shRNA系統的原因 48 4.6 多啟動子表現多個shRNA系統的抑制力 49 4.7 未來展望 50 參考文獻…………………………………………52 | |
dc.language.iso | zh-TW | |
dc.title | 以雙股腺相關病毒投予干擾核醣核酸基因治療的有效性及毒性研究 | zh_TW |
dc.title | Efficacy and Toxicity Study of dsAAV-delivered RNAi Therapy | en |
dc.type | Thesis | |
dc.date.schoolyear | 95-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 楊雅倩(Ya-Chien Yang),林淑華(Shu-Wha Lin),吳慧琳(Hui-Lin Wu) | |
dc.subject.keyword | 干擾RNA,慢性B型肝炎,雙股腺相關病毒,毒性,飽和miRNA路徑,多shRNA表達系統, | zh_TW |
dc.subject.keyword | RNA interference,chronic hepatitis B,double-stranded adeno-associated virus,toxicity,saturation,multiple shRNA system, | en |
dc.relation.page | 87 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2007-07-31 | |
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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