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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 楊宏志(Hung-Chih Yang) | |
dc.contributor.author | Chun-Han Huang | en |
dc.contributor.author | 黃駿涵 | zh_TW |
dc.date.accessioned | 2021-06-17T01:32:29Z | - |
dc.date.available | 2023-08-01 | |
dc.date.copyright | 2018-08-01 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-08-02 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/67444 | - |
dc.description.abstract | B型肝炎病毒隸屬於肝病毒科(Hepadnaviridae),而在感染進入人體肝細胞後,病毒原本帶有的鬆弛環狀DNA (relaxed circular DNA, rcDNA)會在細胞核中轉變成相對穩定的共價閉合環狀DNA (covalently closed circular DNA, cccDNA)。cccDNA的去除是治癒慢性B型肝炎感染的關鍵,但是目前cccDNA的確切形成機制依舊不明。由於rcDNA上有許多結構與需要被修復的受損DNA類似,目前普遍推斷cccDNA的形成過程會有DNA修復機制的酵素參與其中。由於rcDNA必需切除其上的多餘片段才會形成完整的cccDNA,我們的目標是去鑑定出究竟是哪一個人類的結構專一DNA內/外切酶 (structure-specific DNA endo/exonuclease)能夠協助cccDNA的形成。根據酵素個別的屬性,我們總共挑選了7個DNA內/外切酶作為候選基因,包括FEN1, EXO1, GEN1, ERCC4, ERCC5, MRE11 和 DNA2。並且,藉由short hairpin DNA (shRNA) knockdown或小分子化學抑制劑(small molecule chemical inhibitors)的方式來測試候選基因是否與cccDNA的合成過程相關。而為了後續與cccDNA相關實驗的操作便利,我們成功建立了一株基於HepG2細胞為背景,並且可表現表面抗原突變之DHBV的DNA的細胞株。此細胞未來除了能應用在shRNA的實驗上,更因為表面抗原缺乏的特性使之能產生相對大量的cccDNA,使其更便於未來cccDNA相關的實驗之中。在抑制劑的部分,不論是使用PTPD去抑制FEN1,或者是用mirin去抑制MRE11的外切酶活性都不會對cccDNA的合成產生顯著影響。在shRNA knockdown方面,雖然仍需後續其他實驗結果佐證,在利用shRNA去降低一系列候選基因的messenger RNA (mRNA)之後,我們發現只有ERCC5 (XPG)的基因表現量下降似乎會導致cccDNA量的減少。若未來能證實ERCC5真的與cccDNA的合成相關,我們將會進一步了解ERCC5到底在cccDNA的合成過程中確切參與哪些步驟及負責哪些部位的切除。 | zh_TW |
dc.description.abstract | Hepatitis B virus belongs to the Hepadnaviridae family. Upon the entry of hepatitis B virus (HBV) into the infected hepatocytes, the viral relaxed circular (rc) DNA genome is transported to the nucleus, where it is converted to covalently closed circular (ccc) DNA. Hepadnaviral cccDNA is the major barrier to a cure of the chronic HBV infection. However, the mechanisms regulating cccDNA formation remain largely unknown. Since the structure of rcDNA is similar to damaged cellular DNA, host DNA repair enzymes have been speculated to catalyze the conversion of rcDNA to cccDNA. Our goal is to identify structure-specific DNA endo/exonuclease involved in the formation of cccDNA. Therefore, by using shRNA knockdown and small molecule inhibitors, we planned to screen a panel of DNA repair host nucleases, including FEN1, EXO1, GEN1, ERCC4, ERCC5, MRE11 and DNA2, and to identify the one involved in the cccDNA formation. First, we generated a new HepG2-derived surface-deficient duck hepatitis B virus (DHBV-1S)-inducible cell line named iDB1SHG.b.p and utilized it to investigate the cccDNA formation efficiency after the knockdown by shRNA. Since the surface protein of DHBV-1S was deficient, iDB1SHG.b.p could generate more cccDNAs that were readily detected by Southern blot analysis. This makes this cell line ideal for the study of cccDNA formation. We initially focused on FEN1, a nuclease responsible for removing the RNA primer during DNA replication and cleaving 5’ overhanging DNA during base excision repair (BER). However, we found that neither FEN1-specific chemical inhibitor, PTPD, nor FEN1 shRNA knockdown could suppress cccDNA formation. Finally, through the shRNA knockdown screening, we found that knocking down of ERCC5 mRNA expression seemed to affect the formation of cccDNA. However, the detailed mechanisms need further investigation. Our research is still on going, and we will try to confirm this result in different aspects. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T01:32:29Z (GMT). No. of bitstreams: 1 ntu-106-R04445107-1.pdf: 6679697 bytes, checksum: 7b9e35a49ca52713af14489607530278 (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 致謝 ii
中文摘要 iii Abstract v 1. Introduction 1 1.1 History and classification of Hepatitis B virus 1 1.2 Epidemiology and limitation of current clinical treatment 2 1.3 Genome structure and organization of HBV 3 1.4 Replication of hepadnaviruses 4 1.4.1Package of pregenomic RNA and synthesis of rcDNA 4 1.4.2 Forming of cccDNA from rcDNA 6 1.5 The difficulties in the HBV research 6 1.6 DHBV is a good model for studying cccDNA formation of hepadnaviruses 8 1.7 The evidence that the DNA repair pathways are associated with the formation of hepadnaviral cccDNA 8 1.7.1 NHEJ is required for cccDNA formation from dsl DNA 8 1.7.2 TDP2 may be involved in the removal of viral DNA polymerase 10 1.7.3 DNA Polymerase κ is the key cellular factor for the formation of HBV cccDNA 11 1.8 Introduction of FEN Superfamily 13 1.9 The potential role of structure-specific DNA nucleases in cccDNA formation 13 2. Specific Aims 15 3. Materials and Methods 16 3.1 Cell Lines and Culture Conditions 16 3.2 DNA Transfection 17 3.3 Plasmids 17 3.4 Establishment of DHBV inducible cell line 19 3.5 Small molecular inhibitors 20 3.6 The shRNA knockdown system 20 3.7 Modified Hirt's extraction method 21 3.8 Cytoplasm and nucleus DNA separating extraction protocol 22 3.9 Southern Blot Analysis 22 3.10 DIG-labeled probe synthesis 24 3.11 Western Blot Analysis 25 3.12 Total RNA extraction 26 3.13 Reverse Transcription 27 3.14 Real-time PCR 27 4. Results 29 4.1 Establishment of a Blasticidin-Resistant DHBV-inducible Stable Cell Line for the Application of shRNA 29 4.2 Validation of the DHBV replication intermediates in iDB1SHG.b.p cells 31 4.3 CccDNA stably existed in DHBV-inducible cells 32 4.4 PTPD Failed to Inhibit the Formation of cccDNA 33 4.5 Mirin Failed to Inhibit the Formation of cccDNA 33 4.6 Co-treatment of PTPD and mirin Failed to Inhibit the Formation of cccDNA 34 4.7 Knockdown of candidate nuclease genes in iDB1SHG.b.p 34 5. Discussion 36 5.1 iDB1SHG.b is an ideal tool for the future research of cccDNA formation 36 5.2 CccDNA is more stable than rcDNA in the cell culture systems 37 5.3 FEN1 and exonuclease activity of MRE11 may not be involved in cccDNA formation 37 5.4 ERCC5 may be the dominant nuclease involved in the cccDNA formation 38 6. Figures 40 Fig 1. The life cycle of HBV. 40 Fig 2. Generation of DHBV-1S inducible HepG2 cell line. 42 Fig 3. Validation of the DHBV DNA species by digestion with restriction enzymes. 44 Fig 4. Detection of DHBV DNA replicative intermediates in cytoplasmic and nuclear compartments. 45 Fig 5. DHBV cccDNA specific PCR. 46 Fig 6. cccDNA is highly stable and remains in cell for days. 47 Fig 7. The effects of PTPD treatment on DHBV replication. 49 Fig 8. The effects of mirin on DHBV replication. 51 Fig 9. The effects of Co-treatment with PTPD and Mirin on DHBV replication in iDBHG-a and iDB1SHG.b.p. 52 Fig 10. Validation of the cccDNA formation efficiency after FEN1 knockdown. 54 Fig 11. Validation of the cccDNA formation efficiency after DNA2, ERCC4 and MRE11 genes knockdown 56 . 56 Fig 12. Validation of the cccDNA formation efficiency after ERCC5 and GEN1 knockdown. 57 Fig 13. Measurement of the cccDNA formation efficiency after ERCC5 transient knockdown. 58 7. Reference 59 8. Supplementary Information 63 Table 1. List of shRNA purchased from RNAi core facility 63 Table 2. Primer of each genes in SybrGreen real-time PCR application 64 | |
dc.language.iso | zh-TW | |
dc.title | 尋找參與肝炎病毒共價閉合環狀DNA形成機制之核酸酶 | zh_TW |
dc.title | To Identify the Host Nuclease Regulating the Formation of Hepadnaviral Covalently-closed-circular DNA | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄧述諄(Shu-Chun Teng),李財坤(Tsai-Kun Li) | |
dc.subject.keyword | B型肝炎病毒,cccDNA,rcDNA,FEN1,ERCC5 (XPG), | zh_TW |
dc.subject.keyword | hepatitis B virus,cccDNA,FEN1,ERCC5 (XPG), | en |
dc.relation.page | 64 | |
dc.identifier.doi | 10.6342/NTU201702429 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-08-03 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 微生物學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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