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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳培哲(Pei-Jer Chen) | |
| dc.contributor.author | Yung-Jiun Chen | en |
| dc.contributor.author | 陳永濬 | zh_TW |
| dc.date.accessioned | 2022-11-23T09:10:51Z | - |
| dc.date.available | 2022-08-11 | |
| dc.date.available | 2022-11-23T09:10:51Z | - |
| dc.date.copyright | 2021-08-31 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-08-11 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79774 | - |
| dc.description.abstract | 臨床觀察中，HBV慢性感染者接受Rituximab藥物治療，體內B細胞大量清除後，有高機率發生HBV再活化，且伴隨肝損傷的症狀，因此我們認為B細胞在控制HBV的慢性感染上，扮演重要的角色。B細胞除了分化成漿細胞產生抗體外，也具有抗原呈現以及分泌細胞激素的能力，B細胞被清除後，可能影響其他免疫細胞的數量及功能，甚至改變肝臟免疫微環境，除此之外，目前已知肝細胞中Smc6蛋白可抑制HBV表現，因此我們推測B細胞清除所導致的免疫微環境改變可能進一步影響肝細胞中Smc6蛋白抑制HBV的能力，進而導致HBV再活化，為證實此推測，本研究將建立B細胞清除導致HBV再活化的小鼠模式。 本實驗使用HBV hydrodynamic injection的CBA小鼠模擬慢性感染，再以anti-CD20 (clone 5D2) 清除小鼠B細胞及合併prednisone觀察HBV再活化。實驗結果中，先初步測試anti-CD20及prednisone劑量，分別以75 μg及2.5 mg/kg並比照臨床治療B細胞淋巴瘤的R-CHOP療程投予藥物。於療程中，未觀察到小鼠血液HBV DNA上升的趨勢。除了檢測血液中HBV DNA，也偵測小鼠肝細胞HBV RNA及HBc蛋白於給藥前後的表現量，也未觀察到上升的趨勢。後續實驗則是透過觀察Smc6蛋白及肝臟免疫細胞族群分析，探討HBV再活化及背後潛在的機制，結果發現給予合併治療的小鼠肝臟Smc6蛋白量相較給藥前有所減少，但在肝臟免疫細胞族群分析並未觀察到明顯的改變。 根據研究結果，難以觀察HBV再活化現象，推測為小鼠無法被HBV再感染，HBV表現上升趨勢太過輕微，難以偵測。在宿主層面，小鼠給予治療後，肝臟免疫細胞組成並無明顯改變，這可能解釋小鼠未發生再活化的原因。但合併治療組別的Smc6給藥後有明顯下降，揭開HBV再活化背後的可能機制。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-23T09:10:51Z (GMT). No. of bitstreams: 1 U0001-1108202112200600.pdf: 5526945 bytes, checksum: 406cc1194496b328a98f88509974db79 (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | "口試委員會審定書.....................................................................i 誌謝...............................................................................ii 中文摘要..........................................................................iii Abstract...........................................................................iv Chapter 1-Introduction..............................................................1 1.1 Hepatitis B virus...............................................................1 1.1.1 HBV epidemiology..............................................................1 1.1.2 HBV life cycle................................................................2 1.1.3 The regulation of HBV replication by HBx and Smc5/6 complex...................3 1.2 Immune response in HBV infection................................................4 1.2.1 Innate immune response to HBV.................................................4 1.2.2 Adaptive immune response to HBV...............................................5 1.2.2.1 T cell immunity in HBV infection............................................6 1.2.2.2 B cell immunity in HBV infection............................................7 1.3 The immune environment in livers................................................7 1.4 Rituximab for B cell depletion therapy..........................................9 1.5 R-CHOP therapy for B cell lymphoma.............................................10 1.6 Rituximab-associated treatment leading to HBV reactivation.....................10 1.7 Hypothesis.....................................................................11 Chapter 2-Material and Methods.....................................................13 2.1 Animal model...................................................................13 2.2 Establishing HBV chronic infection mice model by hydrodynamic injection........13 2.3 Anti-CD20 and prednisone therapy...............................................14 2.4 The measurement of HBsAg in mice serum.........................................14 2.5 The Quantification of HBV DNA in murine serum..................................15 2.6 Isolation of murine peripheral blood mononuclear cells (PBMC)..................15 2.7 Mice hepatectomy...............................................................16 2.8 RNA extraction and northern blotting for HBV RNA from resected murine livers...16 2.9 Protein extraction from resected murine livers and western blotting............17 2.10 The quantification of HBV plasmid in murine livers............................17 2.11 The isolation of NPC in murine livers.........................................18 2.12 Flow cytometry staining and analysis..........................................19 Chapter 3-Result...................................................................21 3.1 The drug dosage test...........................................................21 3.1.1 The efficiency of anti-CD20 (clone 5D2) in different dosages.................21 3.1.2 The effective dosage of prednisone...........................................21 3.2 The combination therapy of anti-CD20 and prednisone to HBV-carried mice........22 3.2.1 Anti-CD20 (clone 5D2) maintained B cells at low level by multiple doses......23 3.2.2 HBV DNA didn’t increase after the treatment..................................24 3.3 Intracellular observation of HBV reactivation..................................24 3.3.1 HBV RNA and HBc expression in murine liver didn’t increase after treatment...25 3.3.2 The amount of Smc6 decreased in one mouse after the combination therapy......26 3.4 The analysis of NPCs after anti-CD20 (clone 5D2) and prednisone treatment......27 Chapter 4-Conclusion and Discussion................................................29 Chapter 5 Figures..................................................................33 Figure 1∣HBV life cycle............................................................33 Figure 2∣Efficiency of B cell depletion by different dosages of anti-CD20 (clone 5D2)...............................................................................34 Figure 3∣Efficiency of prednisone with different dosages...........................35 Figure 4∣B cells, CD4 T cells and CD8 T cells flow cytometry analysis before and after different dosages of prednisone treatment....................................36 Figure 5∣The flow diagram of anti-CD20 and prednisone treatment....................37 Figure 6∣B cells, CD4 and CD8 T cells analysis after multiple doses of anti-CD20 and prednisone treatment...............................................................39 Figure 7∣HBV DNA in murine serum during anti-CD20 and prednisone treatment.........40 Figure 8∣The experimental design of intracellular observation of HBV reactivation..41 Figure 9∣Northern blotting for HBV RNA expression before and after anti-CD20 and prednisone treatment...............................................................43 Figure 10∣Western blotting for HBc before and after anti-CD20 and prednisone treatment..........................................................................44 Figure 11∣Western blotting for Smc6 before and after anti-CD20 and prednisone treatment..........................................................................45 Figure 12∣The number of immune cells in murine livers and spleens..................46 Supplemental Figure 1∣Dot plot results of B cell depletion by different dosages of anti-CD20 (clone 5D2)..............................................................48 Supplemental Figure 2∣The body weight of mice......................................49 Supplemental Figure 3∣HBsAg in murine serum during anti-CD20 and prednisone treatment..........................................................................50 Supplemental Figure 4∣HBV DNA and HBsAg in the serum of one mouse that possibly got HBV reactivation...................................................................51 Supplemental Figure 5∣The speculation of HBV reactivation in human or hydrodynamic injection mice system..............................................................52 Supplemental Figure 6∣The dot plot results of lymphocytes in livers and spleens....55 Supplemental Figure 7∣The dot plot results of myeloid cells in livers and spleens..58 Chapter 6 Tables...................................................................59 Table 1∣The normalization of HBV RNA in murine livers..............................59 Table 2∣The yield rate of NPCs and splenocytes in each mouse.......................60 References.........................................................................61" | |
| dc.language.iso | en | |
| dc.subject | 肝臟免疫微環境 | zh_TW |
| dc.subject | B細胞清除 | zh_TW |
| dc.subject | B型肝炎再活化 | zh_TW |
| dc.subject | Smc6 | zh_TW |
| dc.subject | B型肝炎病毒 | zh_TW |
| dc.subject | HBV reactivation | en |
| dc.subject | Smc6 | en |
| dc.subject | liver immune microenvironment | en |
| dc.subject | Hepatitis B virus (HBV) | en |
| dc.subject | B cell depletion | en |
| dc.title | 建立B細胞清除誘導B型肝炎病毒再活化的小鼠模式 | zh_TW |
| dc.title | Establishment of a B cell depletion induced HBV reactivation mouse model | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 許秉寧(Hsin-Tsai Liu),楊宏志(Chih-Yang Tseng) | |
| dc.subject.keyword | B型肝炎病毒,B細胞清除,B型肝炎再活化,Smc6,肝臟免疫微環境, | zh_TW |
| dc.subject.keyword | Hepatitis B virus (HBV),B cell depletion,HBV reactivation,Smc6,liver immune microenvironment, | en |
| dc.relation.page | 64 | |
| dc.identifier.doi | 10.6342/NTU202102273 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2021-08-11 | |
| 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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