虎杖根部乙醇萃取物對 EB 病毒潛伏期膜蛋白質 1 功能的影響

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標題:	虎杖根部乙醇萃取物對 EB 病毒潛伏期膜蛋白質 1 功能的影響 Effects of ethanolic extract from Polygonum cuspidatum root on the functions of Epstein-Barr virus latent membrane protein 1
作者:	陳怡頻
貢獻者:	林翠品嘉南藥理科技大學：營養與保健科技研究所
關鍵字:	潛伏期蛋白質 1 虎杖 Epstein-Barr 病毒 Polygonum cuspidatum Latent membrane protein 1 EBV
日期:	2009
上傳時間:	2010-04-15 16:47:47 (UTC+8)
摘要:	EB 病毒是普遍存在於人類的疱疹病毒，與許多惡性腫瘤有密切的相關性。感染 EB 病毒於潛伏期時，病毒編碼出具有致癌基因特性的潛伏期膜蛋白質 1 (LMP1)。在東亞地區傳統藥用植物虎杖常被應用在炎症疾病、肝炎和腫瘤的治療。本研究探討虎杖根部乙醇萃取物 (PcE) 對於含有 EB 病毒的細胞有何影響。實驗以 MTT 方法測定 B95-8 細胞的存活率。免疫墨點法測定 EB 病毒溶裂期蛋白質 Rta 及潛伏期蛋白質 LMP1 的表現。螢光素酶活性分析探討 PcE 對 LMP1 啟動子轉錄活性。以間接免疫螢光法觀察細胞核內 NF-B 的表現量。使用螢光 DNA 結合染劑，DAPI將細胞核染色體染色後，由螢光顯微鏡觀察細胞核形態當成細胞凋亡的一個指標。進一步以慧星試驗分析 DNA 分解斷裂的片段，再以流式細胞儀定量細胞凋亡百分比。結果發現 PcE (25-100 g/ml) 對 B95-8 細胞的存活率具有時間及劑量依存效應，CC50 為 76 g/ml。PcE 濃度 50 g/ml時可有效抑制 Rta 及 LMP1 的表現，並且抑制 LMP1 啟動子轉錄活性，也讓細胞核內的 NF-B 量明顯減少。由螢光顯微鏡觀察細胞核也出現典型凋亡特徵及細胞 DNA 斷片增加的趨勢。在細胞經 PcE 處理 12 及 24 小時後早期及晚期凋亡比率隨濃度增加而提高，sub-G1的細胞凋亡百分比約增加五倍。研究結果指出，PcE 藉由抑制 LMP1 表現及降低細胞核內 NF-B 表現導致 EB 病毒腫瘤細胞凋亡，所以 PcE 有潛力開發為對抗 EB 病毒腫瘤細胞的治療藥物。 Epstein-Barr virus (EBV) is a gamma herpesvirus that is associated with several malignant diseases. EBV-encoded latent membrane 1 (LMP1) is an oncogenic properties, which is expressed during viral latency. Polygonum cuspidatum Sieb. et Zucc. (P. cuspidatum) has been used as a folk medicine for treating inflammatory disease, hepatitis and tumors in East Asia. This study investigates the effects of ethanolic extract from Polygonum cuspidatum root (PcE) on the functions of EBV latent membrane protein 1. This investigation used MTT assay to determine the viabilities of B95-8 cells. Immunoblot was performed to examine the expression of Rta and LMP1, two EBV-encoded proteins. Transient transfection analysis was used to assess the transcription of EBV LMP1. Indirect immunofluorescence assay was performed to examine the expression of activated NF-B (p65). 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. My study found that the viability of B95-8 cells decreases when the cells were treated with PcE (25-100 g/ml) in a time- and dose-dependent manner. The CC50 of the PcE is 76 g/ml. The PcE at concentrations of 50 g/ml not only inhibits the transcription of the Rta and LMP1 genes of LMP1 promoter, but also depletes NF-B in the nuclei of B95-8 cells. Fluorescence microscopy showed apoptosis and DNA strand breaks of the cells. The cells that exhibit early and late apoptosis increase at 12 and 24 hr after exposure to PcE. The sub-G1 apoptosis percentage increase by around fivefold. This study finds that PcE promotes apoptosis due to its ability to inhibit the expression of latent membrane protein 1 and reduced NF-B expression in nuclei. These results indicated that PcE may be a useful therapeutic drug to kill EBV-positive cells.
關聯:	校內校外均不公開，學年度：97， 85 頁
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