EB病毒再活化進入溶裂期感染已被認為與鼻咽癌有密切相關。但其在癌症發展中所扮演的角色目前仍不清楚。我們選擇使用EB病毒在鼻咽癌細胞中再活化的體外模式來探討此課題。然而，過去用於誘發EB病毒再活化的藥物，往往過度活化細胞中的訊息傳遞路徑或引起細胞凋亡，反而可能掩蓋EB病毒溶裂期產物對宿主細胞所造成的影響。為避免這些活化或毒殺細胞的副作用，EB病毒的再活化也可以經由外源表現一個重要的病毒前早期蛋白質Zta而被誘發。Zta為一個轉活化因子，可以啟動並促進下游的病毒溶裂期基因接續表現。
本篇研究中，我們建立了以doxycycline誘導Zta表現的方式，來啟動EB病毒在鼻咽癌細胞中再活化的模式。首先我們利用帶有綠色螢光基因的EB病毒感染可用doxycycline誘導Zta表現的鼻咽癌細胞株HONE-tetonZ後，再以限數稀釋的方式挑出單系細胞群落，透過觀察綠色螢光並以原位雜交反應偵測EB病毒小RNA的表現，挑選確認成功受到EB病毒感染的細胞，以doxycycline刺激該細胞表現Zta後，確實啟動EB病毒再活化，依序表現出溶裂期蛋白質，包括前早期抗原Zta與Rta，早期抗原BMRF1與BHRF1，以及晚期抗原VCA與MA。Real-time PCR的結果顯示，以doxycycline處理後，細胞中EB病毒的DNA量明顯增加，表示病毒大量的進行溶裂期DNA複製。此外，細胞經doxycycline刺激後，其細胞培養液中含有較多的細胞外病毒DNA，以及具有感染能力之病毒顆粒，顯示在我們的細胞模式中，EB病毒可以完成整個溶裂期的過程。MTT實驗結果也證明，相較於過去刺激病毒溶裂期的藥物， doxycycline對於細胞存活率的影響甚小。 既然doxycycline具有毒性小、副作用少的優點，這個能讓EB病毒有效率再活化的細胞模式將有助於探討病毒溶裂期感染如何影響鼻咽癌細胞或促成此癌症的發展。

Reactivation of Epstein-Barr virus (EBV) into the lytic cycle has been associated with nasopharyngeal carcinoma (NPC), but the roles of EBV lytic infection in the cancer development are unclear. To answer this question, we choose to use an in vitro model of EBV reactivation in NPC cells. However, previous studies stimulated EBV reactivation by using reagents that either over-activated cellular signaling pathways or caused cell death, which may have largely masked the effects of EBV lytic products on the host cells. Bypassing the lytic stimulators with cytoactive or cytotoxic side effects, EBV reactivation can also be induced by ectopic expression of Zta, an EBV immediate-early transactivator that essentially initiates and promotes expression cascade of downstream lytic genes. In this study, we established an EBV reactivation model in a NPC cell line where Zta expression is driven by a doxycycline-inducible promoter. First, we infected the doxycycline-inducible, Zta-expressing NPC cell line HONE-tetonZ with a recombinant EBV carrying an expression cassette of enhanced green fluorescence protein (EGFP) in the viral genome. The EGFP-positive cells were cloned by limiting dilution and EBV infection in the cells was re-confirmed by in situ hybridization detecting EBV-encoded small RNAs. Doxycycline treatment of the cells induced Zta, followed by serial expression of other EBV lytic proteins including the immediate-early antigens (Zta and Rta), two early antigens (BMRF1 and BHRF1), and two late antigens (virus capsid protein and membrane protein). Real-time PCR assay also showed significant increase of intracellular EBV DNA after doxycycline treatment, indicating viral DNA amplification in the lytic cycle. Moreover, culture supernatants of the doxycycline-treated cells contained more extracellular viral DNA and infectious virions, indicating completion of EBV lytic cycle in our cell model. Results from MTT assay showed that doxycycline had little effect on cell viability, compared with previous lytic stimulators. Considering the low toxicity and limited side effects of doxycycline, this cell model of efficient EBV reactivation will be useful to explore how the lytic infection affects NPC cells or contributes to NPC development.

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