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請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80804
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dc.contributor.advisor陳美如(Mei-Ru Chen)
dc.contributor.authorHsiang-Chi Fanen
dc.contributor.author范翔淇zh_TW
dc.date.accessioned2022-11-24T03:17:11Z-
dc.date.available2021-11-05
dc.date.available2022-11-24T03:17:11Z-
dc.date.copyright2021-11-05
dc.date.issued2021
dc.date.submitted2021-10-06
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80804-
dc.description.abstractEB病毒 (Epstein-Barr virus) 感染了全球 90% 以上的人口。初次感染後,EBV 在宿主中潛伏,並可能會定期重新激活以產生子代病毒。 EBV 與各種淋巴瘤和上皮腫瘤有關,包括鼻咽癌和胃癌。血清學研究發現,針對 EBV 溶裂期蛋白質的高量抗體或 EBV 的反覆再激活與鼻咽癌的風險相關。在之前的研究中發現包括 BALF3 末端酶、BGLF5 DNA酶和 BGLF4 蛋白激酶在內的溶裂期蛋白會誘導基因組不穩定。另外,實驗室先前的研究證明 BGLF4 在 NPC 細胞中的重複表達誘導了核纖層的變形,並促進細胞通過小於細胞核大小的狹小孔洞。在本論文中,我們檢查了重複表達 BGLF4 對 lamin A/C、磷酸化核纖層完整性、DNA損傷和修復的訊號以及 NPC-TW01 細胞微核 (micronucleus) 形成的累積性的影響。通過使用去氧羥四環素 (doxycycline) 誘導系統的細胞株,我們發現 BGLF4 的重複表達降低了細胞生長速度。重複表達 BGLF4 三代(P3 ) 後,在共焦成像中觀察到細胞損傷指標γ-H2AX 焦點的數量顯著增加,而去活性的BGLF4 (K102I) 組則無看到此現象。雖然 BGLF4 和 γ-H2AX 訊號在 P5 細胞中的表達較低,但累積的損傷可能導致 BGLF4 表達細胞的負選擇,此外,核椎板突出的細胞增加且γ-H2AX信號在突出部位共位。然而,在疝氣狀突出處 (herniation site)未檢測到DNA損傷修復因子53BP1,推測細胞 DNA 損傷修復訊號可能受到核纖層變形所抑制,有待後續進一步釐清。此外,我們在胃癌細胞 AGS 中也試圖建立了去氧羥四環素誘導系統,以進一步檢查 BGLF4 重複表達的累積效應。而BGLF4對EBV表皮細胞癌症的致病機轉仍有待進一步研究探討。zh_TW
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dc.description.tableofcontents論文口試委員審定書 I 致謝 II 中文摘要 III ABSTRACT IV CONTENTS V 1. INTRODUCTION 1 1.1. Epstein-Barr virus (EBV) 1 1.1.1. Classification and Characterization of EBV 1 1.1.2. EBV latency and associated diseases 1 1.2. Conserved Herpes protein kinase (CHPK) 3 1.2.1 Characterization of CHPK 3 1.2.2 EBV protein kinase BGLF4 4 1.2.3 EBV reactivation and tumorigenic function of lytic proteins 4 1.3. The nuclear envelope (NE) 5 1.3.1 The structure and function of the nuclear envelope 5 1.3.2 The importance of the nuclear envelope integrity 6 1.4. Genome Instability (GI) 7 1.4.1 Mutation and repair 7 1.4.2 Infectious agents cause GI 8 1.4.3 EBV reactivation and genome instability 8 1.5. Specific aim 9 2. MATERIALS METHODS 11 2.1. Cell culture 11 2.2. Construction of pLenti4-GFP-BGLF4 plasmid 12 2.3. Western blot analysis 13 2.4. Immunofluorescence assay (IFA) and confocal microscopy 14 2.5. Statistical analyses 15 2.6. Establishment of Tet-on BGLF4 / BGLF4K102I AGS cells 15 3. RESULTS 17 3.1. Repetitive induction of BGLF4 in TW01 cells retarded cell growth. 17 3.2. Repetitive induction of BGLF4 in TW01 cells increased cells with herniation and DNA damage 18 3.3. DNA damage repair was not detected after repetitive induction of BGLF4 in TW01 cells 20 3.4. Repetitive induction of BGLF4 in TW01 cells increased MN formation in P3 but not P5 21 3.5. GFP-BGLF4 and GFP-BGKD expressed after doxycycline induction in Tet-on BGLF4 AGS cells 22 4. DISCUSSION 23 5. FIGURES 28 Fig. 1. Tet-on TW01 cells express BGLF4 after doxycycline induction. 28 Fig. 2. Repetitive induction of BGLF4 expression induces lamin A/C phosphorylation and retards cell growth. 30 Fig. 3. BGLF4 was detected after Dox-off 24-hour but not 96-hour recovery in P3 R and P5 R cells. 32 Fig. 4. Repetitive induction of BGLF4 in BGLF4-2 P3 cells shows the increase of cells with herniation. 34 Fig. 5. Repetitive induction of BGLF4 in BGLF4-2 P5 cells shows a slight increase in herniation. 36 Fig. 6. DNA damage was assessed by γ-H2AX foci counting in individual cells after repetitive induction. 38 Fig. 7. 53BP1 signals were rarely detected at the herniation sites in repetitive expressed BGLF4 P3 cells. 40 Fig. 8. Repetitive expression of BGLF4 increased MN formation in P3 R BGLF4-2. 42 Fig. 9. Repetitive expression of BGLF4 increased MN formation in P5 R BGLF4-2. 44 Fig. 10. Establishment of Tet-on GFP-BGLF4 in AGS cells. 46 6. REFERENCES 47
dc.language.isoen
dc.subjectBGLF4蛋白zh_TW
dc.subjectEB病毒zh_TW
dc.subject基因組不穩定性zh_TW
dc.subjectBGLF4en
dc.subjectEBVen
dc.title探討重複表現EB病毒BGLF4蛋白對於核纖層完整性和基因組不穩定性之影響zh_TW
dc.titleEffects of repetitive expression of EBV BGLF4 on nuclear lamina integrity and genome instabilityen
dc.date.schoolyear109-2
dc.description.degree碩士
dc.contributor.oralexamcommittee李明學(Hsin-Tsai Liu),曾紀綱(Chih-Yang Tseng),李重霈
dc.subject.keyword基因組不穩定性,EB病毒,BGLF4蛋白,zh_TW
dc.subject.keywordEBV,BGLF4,en
dc.relation.page51
dc.identifier.doi10.6342/NTU202103556
dc.rights.note同意授權(限校園內公開)
dc.date.accepted2021-10-06
dc.contributor.author-college醫學院zh_TW
dc.contributor.author-dept微生物學研究所zh_TW
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