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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 顏伯勳(Bo-Shiun Yan) | |
dc.contributor.advisor | 顏伯勳(Bo-Shiun Yan | bsyan@ntu.edu.tw | ), | |
dc.contributor.author | Yi-Chun Kao | en |
dc.contributor.author | 高儀均 | zh_TW |
dc.date.accessioned | 2023-03-19T22:29:19Z | - |
dc.date.copyright | 2022-10-05 | |
dc.date.issued | 2022 | |
dc.date.submitted | 2022-08-29 | |
dc.identifier.citation | [1]Franco-Paredes C, Marcos LA, Henao-Martínez AF, et al. Cutaneous Mycobacterial Infections. Clin Microbiol Rev 2018;32(1). [2]Falkinham JOP. Environmental Sources of Nontuberculous Mycobacteria. Clinics in Chest Medicine 2015;36(1):35-41. [3]Shao-Tsung Huang R-MH, Suh-Ling Hwang, Chia-Jung Chiang. Non-tuberculosis Mycobacterium and Healthcare-associated Infection. 感染控制雜誌 2010;20卷5期:306 - 317. [4]Henry MT, Inamdar L, O'Riordain D, et al. Nontuberculous mycobacteria in non‐HIV patients: epidemiology, treatment and response. 2004;23(5):741-746. [5]Koh WJ, Kwon OJ, Jeon K, et al. Clinical significance of nontuberculous mycobacteria isolated from respiratory specimens in Korea. Chest 2006;129(2):341-348. [6]van Ingen J, Bendien SA, de Lange WC, et al. Clinical relevance of non-tuberculous mycobacteria isolated in the Nijmegen-Arnhem region, The Netherlands. Thorax 2009;64(6):502-6. [7]Patrias K, Wendling D. Citing Medicine. Department of Health and Human Services, National Institutes of Health, US …; 2007. [8]Wu M-F, Shu C-C, Wang J-Y, et al. NLRP3 inflammasome is attenuated in patients with Mycobacterium avium complex lung disease and correlated with decreased interleukin-1β response and host susceptibility. Scientific Reports 2019;9(1):12534. [9]Zheng D, Liwinski T, Elinav E. Inflammasome activation and regulation: toward a better understanding of complex mechanisms. Cell Discovery 2020;6(1):36. [10]Lou J, Wang Y, Zhang Z, et al. MiR-20b inhibits mycobacterium tuberculosis induced inflammation in the lung of mice through targeting NLRP3. Experimental Cell Research 2017;358(2):120-128. [11]Chen C-C, Tsai S-H, Lu C-C, et al. Activation of an NLRP3 Inflammasome Restricts Mycobacterium kansasii Infection. PLOS ONE 2012;7(4):e36292. [12]Swanson KV, Deng M, Ting JPY. The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nature Reviews Immunology 2019;19(8):477-489. [13]Schett G, Dayer J-M, Manger B. Interleukin-1 function and role in rheumatic disease. Nature Reviews Rheumatology 2016;12(1):14-24. [14]Dinarello CA. Interleukin 1 and interleukin 18 as mediators of inflammation and the aging process. The American Journal of Clinical Nutrition 2006;83(2):447S-455S. [15]Uddin F, Rudin CM, Sen T. CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future. 2020;10. [16]Health NIo. Help me understand genetics: Genomic research. 2020. [17]Lai CC, Tan CK, Chou CH, et al. Increasing incidence of nontuberculous mycobacteria, Taiwan, 2000-2008. Emerg Infect Dis 2010;16(2):294-6. [18]Huang H-L, Cheng M-H, Lu P-L, et al. Epidemiology and Predictors of NTM Pulmonary Infection in Taiwan - a Retrospective, Five-Year Multicenter Study. Scientific Reports 2017;7(1):16300. [19]Shu C-C, Lee C-H, Hsu C-L, et al. Clinical Characteristics and Prognosis of Nontuberculous Mycobacterial Lung Disease with Different Radiographic Patterns. Lung 2011;189(6):467-474. [20]Duan J, Wainwright MS, Comeron JM, et al. Synonymous mutations in the human dopamine receptor D2 (DRD2) affect mRNA stability and synthesis of the receptor. Human Molecular Genetics 2003;12(3):205-216. [21]Karthi S, Rajeshwari M, Francis A, et al. 3′-UTR SNP rs2229611 in G6PC1 affects mRNA stability, expression and Glycogen Storage Disease type-Ia risk. Clinica Chimica Acta 2017;471:46-54. [22]Shatoff E, Bundschuh R. Single nucleotide polymorphisms affect RNA-protein interactions at a distance through modulation of RNA secondary structures. PLOS Computational Biology 2020;16(5):e1007852. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/84857 | - |
dc.description.abstract | 分枝桿菌屬(Mycobacterium)為可以被酸性染劑染色的耐酸菌,包含兩種會造成嚴重疾病的菌種:肺結核分枝桿菌(Mycobacterium tuberculosis)以及痲瘋分枝桿菌(Mycobacterium leprae)。除此之外,還有一些分枝桿菌也會造成臨床的症狀,並被統稱為非結核分枝桿菌(nontuberculous mycobacteria, NTM)。傳統上,人們認為感染NTM所引發的疾病只會發生在少數免疫缺乏的病患身上。然而,近十年來,NTM所造成的肺部疾病(nontuberculous mycobacterial lung disease, NTM-LD)在免疫力正常的族群中,發生率及盛行率皆有增加的趨勢。會有這樣的現象除了微生物學的檢驗技術進步之外,也代表著有些免疫功能正常的患者中,可能帶有其他的風險因子,讓宿主對於疾病具有易感性(susceptibility)。然而,宿主對NTM-LD易感性的機制目前仍舊不清楚。在東亞以及美國地區,最常見會造成NTM-LD的菌種為Mycobacterium avium complex(MAC)。而在宿主受到MAC感染時,會引發體內的巨噬細胞和單核球產生發炎體複合物。發炎體複合物包含不同的感測器(sensor),包括NLRP3,當細胞偵測到病原體相關分子樣式(PAMPs)時NLRP3會被活化。此外,由NLRP3所組成的發炎體已被證實,對於預防分枝桿菌的感染扮演重要的角色。先前的研究中發現,當NLRP3發炎體的表現減少會減弱巨噬細胞的免疫反應。同時也透過分析找到NLRP3基因上數個與MAC-LD有相關性的單一核苷酸多型性(SNP)位點,而rs34298354位點與女性MAC-LD病人群體具有顯著的相關性。為了探討此位點和宿主對於MAC感染的易感性之角色,我們首先在 HEK293T 細胞中表現rs34298354位點有不同等位基因的NLRP3蛋白質。結果顯示,在此位點為T等位基因的 NLRP3 蛋白質表現量有減少的情形。此外,在此位點為T等位基因的 NLRP3基因所轉錄的 RNA顯示出較低的穩定性,這說明NLRP3的rs34298354位點在RNA穩定性中非常重要。為了進一步研究 NLRP3 蛋白質表現量和巨噬細胞功能的相關性,目前正以 CRISPR-Cas9 基因組編輯系統建立在此位點具有兩個等位基因的 THP1 單核細胞白血病細胞株。同時,目前已經以同一系統成功建立在此位點具有兩個等位基因T的 HEK293T 細胞株。藉由建立的這些HEK293T 細胞株,驗證了 NLRP3 基因的 rs34298354 位點確實會影響 RNA 的穩定性和蛋白質的表現量。 | zh_TW |
dc.description.abstract | Mycobacteria are acid-fast bacteria, including Mycobacterium tuberculosis and Mycobacterium leprae, both associated with serious diseases. In addition to those, nontuberculous mycobacteria (NTM) are another group of mycobacteria that cause clinical diseases. Traditionally, it has been thought that NTM infection-caused diseases only happened in immunocompromised people. However, the incidence and prevalence of NTM-lung disease (LD) increased in the last ten years in patients with normal immune systems. Besides the advancement of inspection techniques for microbiology, this increase is also attributed to other risk factors in patients without immune dysfunction. However, the mechanism of the host susceptibility to NTM-LD still remains unclear. Mycobacterium avium complex (MAC) is the most common pathogen associated with NTM-LD in the East Asia and the United States, and inflammasome complex is found to assemble during MAC infection in macrophages and monocytes. Inflammasome complex contains a variety of sensors, including NLRP3, which is induced when pathogen-associated molecular patterns (PAMPs) are detected. Besides, NLRP3-inflammasome plays an important role for preventing mycobacterial infection. It has been demonstrated that impaired expression of NLRP3-inflammasome in macrophages weakens inflammatory responses and several single nucleotide polymorphisms (SNPs) of NLRP3 gene are associated with the MAC-LD. Moreover, the SNP rs34298354 of NLRP3 is significantly associated with MAC-LD in female patients. To investigate the role of the SNP in host susceptibility to MAC infection, NLRP3 protein with different alleles at the SNP rs34298354 was over-expressed in HEK293T cells. The results revealed that the protein level of NLRP3 with T allele at the SNP rs34298354 was decreased. Besides, the transcript from NLRP3 gene with T allele at the SNP rs34298354 showed a significantly lower stability. It suggests that the SNP rs34298354 of NLRP3 plays an important role in RNA stability. To further investigate the association of NLRP3 protein level and macrophage functions, THP1 monocytic leukemia cell lines with two alleles at the SNP rs34298354 have being established via a CRISPR-Cas9 genome editing system. In parallel, HEK293T cell lines with two alleles at the SNP rs34298354 have being established successfully via the same system. Using the generated HEK293T cell lines, it has been validated that the SNP rs34298354 of NLRP3 has molecular effect on RNA stability and protein level. | en |
dc.description.provenance | Made available in DSpace on 2023-03-19T22:29:19Z (GMT). No. of bitstreams: 1 U0001-2908202213563700.pdf: 3119688 bytes, checksum: 3255a0c592f3d1e9c0d1ab7f55442a89 (MD5) Previous issue date: 2022 | en |
dc.description.tableofcontents | 誌謝 i 摘要 ii Abstract iv 第一章、研究背景與目的 1 1-1分枝桿菌(Mycobacterium) 1 1-2 非結核分枝桿菌(NTM) 1 1-3基因易感性(gene susceptibility) 2 1-4 單一核苷酸多型性(single nucleotide polymorphisms, SNP) 3 1-5 發炎體(inflammasome) 3 1-6 NLRP3發炎體(N: NOD-like or nucleotide-binding domain, LR: leucine-rich repeat, P: pyrin domain-containing protein) 4 1-6.1 NLRP3發炎體的組成 4 1-6.2 細胞激素(cytokine)釋放 5 1-6.3 細胞焦亡(pyroptosis) 6 1-7研究目標 6 第二章、實驗材料與方法 7 2-1細胞株和細胞培養 7 2-2 載體 7 2-2.1 pcDNA3.1-CMV載體 7 2-2.2 LentiCRISPRv2載體 7 2-2.3 pHAGE-TRE-mDsRed-Linker-IRES-dLNGFR載體 8 2-2.4 GenBulider Plus Cloning Kit(Genscript) 8 2-3 細胞轉染(Transfection) 8 2-4 西方墨點法(Western blot) 8 2-5 以放線菌素D(Actinomycin D)進行RNA穩定性(stability)的分析 9 2-6 RNA準備以及mRNA的定量 9 2-7 慢病毒生產(Lentivirus production) 10 2-8 慢病毒轉染(Lentivirus transduction)和細胞克隆 10 2-9 基因體DNA(genomic DNA)的萃取與純化 10 2-10 統計分析和圖表呈現 11 第三章、實驗結果 12 3-1 分析rs34298354位點對NLRP3 protein的影響 12 3-1.1 質體DNA的製備 12 3-1.2 質體DNA蛋白質表現的確認 12 3-1.3 置換質體DNA中NLRP3序列的rs34298354位點 13 3-1.4 以西方墨點法分析rs34298354位點的不同是否影響NLRP3的表現量 13 3-1.5 製備新的質體以提供西方墨點法分析時另一種校正基準 14 3-1.6 使用新製備的質體再次進行西方墨點法的分析 14 3-1.7 分析rs34298354位點的不同是否會影響NLRP3 RNA的穩定度 15 3-2 置換內源性NLRP3基因體DNA的rs34298354位點 16 3-2.1 透過CRISPR-Cas9系統進行基因體DNA編輯 16 3-2.2 製備CRISPR-Cas9系統所需材料 17 3-2.3 以HEK293T細胞株進行CRISPR-Cas9系統的測試 18 3-2.4 運用雙股DNA形式的Donor DNA片段於CRISPR-Cas9系統 19 3-2.5 挑選rs34298354位點置換完成的HEK293T單一細胞株 19 3-2.6 以單一細胞株進行NLRP3基因的RNA穩定度測試 20 3-2.7 透過CRISPR-Cas9系統進行單核白血球細胞株THP-1的基因編輯 21 第四章、結論與討論 23 參考文獻 27 圖附錄 29 附錄 44 | |
dc.language.iso | zh-TW | |
dc.title | 一個非結核分枝桿菌肺病相關的NLRP3基因單一核苷酸多型性位點之生化作用機制 | zh_TW |
dc.title | Biochemical mechanism of an NLRP3 SNP associated with nontuberculous mycobacterial lung disease | en |
dc.type | Thesis | |
dc.date.schoolyear | 110-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 詹世鵬(Shih-Peng Chan),樹金忠(Chin-Chung Shu) | |
dc.subject.keyword | 非結核分枝桿菌,宿主易感性,NLRP3發炎體,單一核苷酸多型性,基因編輯, | zh_TW |
dc.subject.keyword | Nontuberculous mycobacteria,Mycobacterium avium complex,host susceptibility,NLRP3 inflammasome,single nucleotide polymorphisms,gene editing, | en |
dc.relation.page | 46 | |
dc.identifier.doi | 10.6342/NTU202202925 | |
dc.rights.note | 同意授權(限校園內公開) | |
dc.date.accepted | 2022-08-29 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
dc.date.embargo-lift | 2022-10-05 | - |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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