- 學位論文
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
Effects of repetitive expression of EBV BGLF4 on nuclear lamina integrity and genome instability
摘要
EB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。
並列摘要
Epstein-Barr virus (EBV) infects more than 90% of the population worldwide. After primary infection, EBV becomes latent in the host and may periodically be reactivated to produce progeny viruses. EBV is associated with various lymphomas and epithelial tumors, including nasopharyngeal carcinoma and gastric cancer. Serological studies have found that high titer antibodies against EBV lytic proteins or recurrent reactivation of EBV correlate with the risk of nasopharyngeal carcinoma. Lytic proteins including BALF3 terminal enzyme, BGLF5 DNase, and BGLF4 protein kinase were found to induce genome instability in previous studies. I also demonstrated that repetitive expression of BGLF4 in NPC cells induced deformation of the nuclear lamina and promoted cell migration through a confined space that is smaller than the size of the nucleus. In this study, I examined the accumulation effects of BGLF4 expression on lamin A/C phosphorylation, nuclear lamina integrity, DNA damage and repair signals, and micronucleus formation on NPC-TW01 cells. By using the doxycycline-inducible cell lines, I found that repetitive expression of BGLF4, but not the kinase-dead control, reduced the cell growth rate, and significantly increased the number of γ-H2AX foci after repetitive expression BGLF4 for third generations (P3) in confocal imaging analysis. The expression of BGLF4 and the γ-H2AX signals were reduced in P5 cells, suggesting accumulating damages may cause a negative selection of BGLF4 expressing cells. Furthermore, cells with nuclear lamina herniation were increased in third generations (P3), coupling with the signals of γ-H2AX at the herniation sites. The DNA damage repair factor, 53BP1, was not detected at the hernia protrusion, suggesting that the cell DNA damage repair signaling may be inhibited by the deformation of the nuclear lamina. In addition, a doxycycline-inducible system was established in Gastric carcinoma cell line AGS for further examination of the accumulation effects of BGLF4 repetitive expression.
參考文獻
延伸閱讀
- 蔡函育（2021）。探討EB病毒產物在不朽化之類淋巴母芽細胞株中透過RLRs路徑對IL-10表現之影響〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU202102668
- Liao, Y. T. (2017). BFLF2蛋白之進核及與BFRF1交互作用序列對於EB病毒顆粒釋出之影響 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU201701734
- 林如愔（2010）。EB病毒膜蛋白LMP2A抑制人類第二型主要組織相容性複合體基因表現之探討〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2010.10588
- Eglinger, S. T., Bahleda, R., Côté, J. F., Terrier, P., Vidaud, D., Cayre, A., Beauchet, A., Anton, N. T., Lacombe, M. J. T., Lemoine, A., Llorca, F. P., Cesne, A. L., & Emile, J. F. (2008). Frequent EGFR Positivity and Overexpression in High-Grade Areas of Human MPNSTs. Sarcoma, 2008(), 114-120. https://doi.org/10.1155/2008/849156
- Ma, D., Ma, C., Gao, M., Li, G., Niu, Z., & Huang, X. (2012). Induction of Cellular Immune Response by DNA Vaccine Coexpressing E. acervulina 3-1E Gene and Mature CHIl-15 Gene. Journal of Parasitology Research, 2012(), 370-376. https://doi.org/10.1155/2012/654279