臺灣博碩士論文加值系統

Epstein-Barr 病毒 (EBV) 是人類的皰疹病毒，與許多人類淋巴組織惡性腫瘤發生有關。先前實驗室發現虎杖乙醇萃取物藉由抑制潛伏膜蛋白質 1 (LMP1) 及減少細胞核 NF-B 表現而促進 EB 病毒腫瘤細胞走上凋亡，所以本研究將探討虎杖乙酸乙酯分離物 F3a 抗 EB 病毒腫瘤細胞的機制。實驗將以 Trypan blue 方法分析虎杖乙酸乙酯分離物 F3a 對 EB 病毒潛伏期第三型腫瘤細胞，Raji 細胞存活的影響；以即時定量 PCR 分析 EB 病毒基因的 mRNA 及 DNA 表現量；以 DCFH-DA 試劑分析活性氧物質 (ROS) 產量；以西方墨點法分析潛伏膜蛋白質1、凋亡相關蛋白質、ERK及 IB表現量；以流式細胞儀分析凋亡細胞數。結果顯示虎杖乙酸乙酯分離物 F3a 會導致 Raji 細胞死亡，其 CC50 為 12.08 g/ml ，也發現濃度 12.5 g/ml F3a 使 Raji 細胞內 EB 病毒的 BRLF1 及 BNLF1 mRNA 表現增加，潛伏膜蛋白質 1 (LMP1) 的蛋白質表現量增加，也會使 Raji 細胞內磷酸化的 ERK 及磷酸化的 IB 降低，細胞內 ROS 增加，細胞凋亡相關蛋白質，裂解的 caspase 及 PARP 產生，凋亡細胞數增加。所以虎杖乙酸乙酯分離物 F3a 具有開發成抗 EB 病毒相關腫瘤藥物的潛能。

Epstein-Barr virus (EBV) is a human herpesvirus; and is associated with several human lymphoid malignancies. Our earlier study found that the ethanolic extract of Polygonum cuspidatum root promotes apoptosis of EBV-positive cells due to its ability to inhibit the expression of latent membrane protein 1 and deplete NF-B in the nucleus. Therefore, the purpose of this study is to investigate the effects of the ethyl acetate fraction F3a from P. cuspidatum on Raji cells. Trypan blue assay was used to determine the viability of Raji cells; real-time quantitative PCR to assess the expression of EBV mRNA and EBV DNA in Raji cells; to measure ROS production using by DCFH-DA kit. The expression of LMP1, apoptotic-related proteins, ERK and IB were determined by western blotting. Flow cytometry analysis was used for the detection of apoptotic cells. Results show that F3a promotes Raji cells death, with a CC50 were of 12.08 g/ml F3a at 12.5 g/ml induces transcription of BRLF1 and BNLF1 and increases the expression of LMP1, which reduces intracellular phospho-ERK and phospho- IB expression. Meanwhile, F3a increases reactive-oxygen species, actived apoptotic-related proteins, caspase 3, cleavage of PARP, and increases the percentage of apoptotic cells. Therefore, F3a can be developed as a therapeutic drug in the treatment of EBV-related tumors.

摘要 Ⅰ
Abstract Ⅱ
目錄 Ⅳ
圖目錄 Ⅷ
表目錄 X
第一章 緒論 1
1-1 與EB病毒感染有關的疾病 1
1-2 EB 病毒的結構與組成 1
1-3 EB 病毒生活史 2
1-4 病毒潛伏期基因及其蛋白質 4
1-5 潛伏期蛋白質 1 (latent membrane protein 1, LMP1) 5
1-6 虎杖 8
1-7 大黃素 (emodin) 抗病毒及抗腫瘤相關研究 8
1-8 本實驗室近年已完成之研究結果 10
1-8-1 虎杖根部乙醇萃取物、乙酸乙酯分化物及分離物 F1、F2、F2a 及 F3 之製備 10
1-8-2 虎杖抗 EB 病毒腫瘤細胞之活性 11
1-9 研究目的 12
第二章 材料方法 13
2-1 使用材料 13
2-2虎杖根部乙酸乙酯分離物 F3a 之製備及純化 17
2-3 細胞培養 18
2-4 培養方法 19
2-5 Trypan blue 試驗 19
2-6 西方墨點法 (Western Blotting) 19
2-6-1 SDS-PAGE 的製備 19
2-6-2蛋白質的製備與電泳 21
2-6-3蛋白質轉漬與抗體反應 23
2-7 EB 病毒 mRNA 的定量 26
2-7-1 抽取細胞內 EB 病毒的 mRNA 26
2-7-2將 mRNA 轉成 cDNA 27
2-7-3 mRNA定量 27
2-8 EB病毒 DNA 的定量 29
2-8-1 抽取細胞內 EB 病毒的 DNA 29
2-8-2 即時定量聚合酵素連鎖反應 (Real-time quantitative polymerase chain reaction) 29
2-8-2-1 EBV DNA 的定量 29
2-8-2-2 -actin DNA 的定量 30
2-9 DCFH-DA 對於細胞 ROS 產生量之測定 31
2-10 流式細胞儀凋亡試驗 31
2-11 統計分析 32
第三章 結果 33
3-1 虎杖根部乙酸乙酯分離物F3a之逆相式高效率液相層析儀分析 33
3-2 虎杖根部乙酸乙酯分離物 F3a 對 Raji 細胞的存活率影響 33
3-3 虎杖根部乙酸乙酯分離物 F3a 對 EB 病毒潛伏期膜蛋白質 1 (LMP1) 表現的影響 33
3-4 虎杖根部乙酸乙酯分離物 F3a 對 Raji 細胞內 EB 病毒溶裂期及潛伏期基因表現的影響 34
3-5 虎杖根部乙酸乙酯分離物 F3a 對 Raji 細胞內 EB 病毒 DNA 複製的影響 35
3-6 虎杖根部乙酸乙酯分離物 F3a 誘發 Raji 細胞內 ROS 產生的影響 36
3-7 虎杖根部乙酸乙酯分離物 F3a 對 Raji 細胞磷酸化 ERK / 非磷酸化 ERK 及磷酸化 IB非磷酸化 IB 蛋白質表現的影響 37
3-8 虎杖根部乙酸乙酯分離物 F3a 對 EB 病毒凋亡相關蛋白質表現的影響 37
3-9 虎杖根部乙酸乙酯分離物 F3a 對Raji 細胞的凋亡影響 38
第四章 討論 40
結論 45
參考文獻 46

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