EB 病毒為人類皰疹病毒，會感染淋巴細胞和上皮細胞。與 EB 病毒感染相關的腫瘤疾病包含巴克氏淋巴瘤 (Burkitt’s lymphoma)、何杰金氏症 (Hodgkin's disease) 及鼻咽癌等疾病。所以本研究探討檸檬桉樹脂乙酸乙酯分劃物及其主要化合物和虎杖根乙酸乙酯分離物是否能藉由抑制潛伏期膜蛋白質 1 表現進而誘發 EB 病毒腫瘤細胞的凋亡。實驗以 MTT 及 AlamarBlue 方法測定 EB 病毒腫瘤細胞株 B95-8 細胞的存活率；西方墨點法測定 EB 病毒溶裂期蛋白質 Rta 及潛伏期蛋白質 LMP1 的表現；轉染及螢光素酶方法測定 LMP1 基因啟動子的轉錄活性；使用螢光 DNA 結合染劑-DAPI 利用顯微鏡觀細胞核型態；利用彗星試驗觀察 DNA 裂解程度，還有使用流式細胞儀來測定早期和晚期凋亡的百分比。檸檬桉樹脂乙酸乙酯中主要化合物，EuEA6 在濃度 23.7 μM 時使細胞 DNA 裂解，對細胞造成基因毒性，因而抑制 EB 病毒溶裂期蛋白質 Rta 及潛伏期蛋白質 LMP1 的表現。虎杖根乙酸乙酯分離物，F3 在濃度 6.3 μg/ml 時細胞 DNA 裂解也增加，對細胞產生基因毒性，而抑制 EB 病毒溶裂期蛋白質，Rta 及潛伏期蛋白質，LMP1 的表現，且細胞經 F3 處理 12 小時就可以增加早期及晚期凋亡百分比。這些結果證明 EuEA6 和 F3 能抑制潛伏期膜蛋白質 1 表現進而誘發 EB 病毒腫瘤細胞的死亡，可以發展成為治療 EB 病毒相關腫瘤的藥物。 Epstein-Barr virus (EBV) is a human herpesvirus, which infect lymphoid and epithelial cells. Infection by this virus is associated with several malignant diseases such as Burkitt’s lymphoma, Hodgkin disease and nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane protein 1 (LMP1) is an oncogenic protein and expressed in EBV-associated tumor cells. This purpose of this study is to investigate whether the ethyl acetate subfractions of Eucalyptus citriodora resin and Polygonum cuspidatum root prevent LMP1 expression and induce apoptosis of EBV positive tumor cells. The viability on EBV positive tumor cells line, B95-8 was determined by MTT and AlamarBlue assay. The expression of Rta and LMP1 was determined by western blotting. Transient transfection analysis was used to assess the transcription of the LMP1 gene, BNF1. Fluorescence DNA-binding dye, DAPI, was used to detect the nuclear chromatin morphology, which is an index of apoptosis. Comet assay was used to assess DNA strand breakage. Percentage of apoptotic cells was measured by flow cytometry. The main compound, EuEA6 isolated from the ethyl acetate fraction of Eucalyptus citriodora resin induce DNA damage and inhibits effectively the expression of Rta and LMP1 damages DNA at the concentration of 23.7 μM. The ethyl acetate subfractions from Polygonum cuspidatum root, F3 inhibit the expression of Rta and LMP1 promote and DNA strand breaks of the cells at 6.3 μg/ml. B95-8 cells treated with F3 exhibit early and late apoptosis increasing at 12 hr. The results suggests that EuEA6 and F3 prevent the LMP1 expression and cause death of EBV positive tumor cells, may be a useful therapeutic drug in EBV positive tumor cells.
