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研究生:葉詠薷
研究生(外文):Yung-Ju Yeh
論文名稱:大量表現在肝臟的微核醣核酸調控C型肝炎病毒複製細胞株病毒基因的表現
論文名稱(外文):Liver-Abundant MicroRNAs Modulate Hepatitis C Virus Gene Expression in HCV Replicon Cells
指導教授:鄭如茜鄭如茜引用關係
指導教授(外文):Ju-Chien Cheng
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:醫學檢驗生物技術學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:53
中文關鍵詞:C型肝炎病毒微核醣核酸
外文關鍵詞:HCVmicroRNA
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微核醣核酸(microRNAs)是一non-coding小片段RNA,透過降解mRNA或抑制mRNA轉譯而抑制標的基因的表現。在植物及非脊椎動物中,miRNA是先天性抗病毒免疫反應佔有重要一環,但仍不清楚miRNA在哺乳動物中是否也扮演類似的角色。C型肝炎病毒(hepatitis C virus, HCV)是隸屬於黃病毒科(Flaviviridae)的唯一成員,其病毒基因體為一條長度約9.6 kb的正股RNA病毒。本研究利用生物資訊分析細胞內miRNA是否會標的在HCV基因體上,並以實驗證明是否調控HCV複製。我們選擇了許多肝臟大量表現的miRNAs,以帶有HCV genome的細胞株進行分析。結果顯示有兩個肝臟大量表現的miRNA過量表現時抑制HCV的表現。為了證明miRNA是標的在HCV基因體上,利用生物資訊預測標的HCV的序列,並且將標的序列進行突變。實驗結果顯示,其中一個miRNA標的HCV序列突變之後使得HCV基因表現回復。另外,miRNA可標的的宿主細胞基因的表現並不影響HCV基因表現。 總結上述的結果,我們發現一個肝臟大量表現的miRNA藉由直接標的在HCV基因體上,進而抑制HCV基因的表現。
MicroRNAs (miRNAs) are small non-coding RNAs which inhibit the expression of target genes by promoting mRNA degradation or inhibiting mRNA translation. Although miRNAs represent a vital component of the innate antiviral immune response in plants and invertebrate animals, it is not clear whether they play the similar roles in the defense of viral infection in mammalian organisms. Hepatitis C virus (HCV) is the only member of the hepacivirus genus in Flaviviridiae. It contains a 9.6 kb single–stranded RNA genome with positive polarity. In this study, bioinformatics analysis was used to investigate whether cellular miRNAs target the HCV genome and modulate HCV replication. Several liver- abundant miRNAs were selected to characterize the effects on HCV subgenomic cells. Our data showed that two liver-abundant miRNAs can down-regulate HCV gene expression in HCV replicon cells. To elucidate whether the miRNA targets on HCV genome, several bioinformatics predicted target sites on HCV genome were mutated by in vitro mutagenesis assay. One mutated target sites significantly reversed the effect of miRNA on HCV gene expression. Knock down the host gene that is targeted by the miRNA showed no effect on expression of HCV. Taken together, we demonstrate a liver abundant miRNA which can down-regulate HCV gene expression by directly targeting on HCV genome.
致謝 I
中文摘要 II
英文摘要 III
第一章 前言 1
第一節 研究背景 1
1.1.1 C型肝炎病毒的介紹 1
1.1.2 微核醣核酸的生合成 3
1.1.3文獻探討細胞內微核醣核酸調控病毒基因表現 5
第二節 研究目的 6
第二章 研究方法 7
第一節 研究設計 7
第二節 研究材料 8
2.2.1 化學藥品 8
2.2.2 儀器 9
2.2.3 引子 10
2.2.4 抗體 11
2.2.5 載體 12
第三節 研究方法 12
2.3.1 細胞株 12
2.3.2 轉染試驗 12
2.3.3 luciferase分析 13
2.3.4 細胞存活率 13
2.3.5 蛋白質檢體製備 14
2.3.6 蛋白質電泳 14
2.3.7 西方墨點法 14
2.3.8 病毒感染 15
2.3.9 免疫螢光染色 15
2.3.10 RNA萃取 16
2.3.11 即時偵測聚合酶連鎖反應 16
2.3.12 製備含有miRNA標的位置之luciferase報導載體 17
2.3.13 Firefly/ Renilla luciferase分析 17
2.3.14 體外突變作用: 18
2.3.15 體外轉錄作用 19
2.3.16 電穿孔法 19
第三章 研究結果 20
第一節 分析細胞內miRNA標的HCV基因體的可能性 20
3.1.1 預測miRNA和標的HCV RNA結合的可能性 20
3.1.2 標的miRNA的選擇 20
3.2.1 以HCV subgenomic細胞株篩選預測的miRNAs 22
3.2.2以HCV full-length replicon細胞株驗證miRNA調控HCV基因表現
的能力 23
3.2.3 miRNA可調控不同genotype HCV基因表現 23
第三節 miRNA標的的HCV RNA影響報導基因表現的篩選 24
3.3.1 預測miRNA標的在HCV RNA的可能性及相關位置 24
3.3.2 miRNA標的序列構築在載體上並篩選報導基因的表現 24
第四節 利用site-directed mutagenesis分析HCV RNA標的序列的突變對HCV基因表現的影響 25
第五節 miRNA標的細胞因子的表現是不影響HCV基因的表現 26
第四章 討論 28
第一節 miRNA標的位置的選擇 28
第二節 miRNA在細胞內的表現 29
第三節 突變株是否影響HCV基因表現 30
第四節 miRNA是否會誘發IFN路徑引起抗病毒反應 30
第五章 結論與建議 32
第一節 結論 32
第二節 建議 32
參考文獻 33
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