| 摘要: | 在我們近年的研究成果已經證明虎杖乙醇萃取物、分離物 F1、F3、白藜蘆醇、白藜蘆醇苷及大黃素能夠抑制丁酸鈉所誘導 EB 病毒極早期基因 BRLF1 及 BZLF1 的轉錄，並且影響 EB 病毒溶裂蛋白質 Rta、Zta 及 EA-D 的表現，進而抑制 EB 病毒複製及病毒顆粒的產生；而且虎杖乙醇萃取物、分離物 F2a、F3及大黃素也能誘發 EB病毒潛伏期第三型的腫瘤細胞，B95-8 細胞凋亡；虎杖乙醇萃取物、分離物 F3能藉由抑制 EB病毒潛伏期膜蛋白質 1 (LMP1)表現及降低細胞核內 NF-B表現，導致B95-8細胞凋 亡。而分離物 F2a 藉由影響 EB 病毒 LMP1、EB 病毒核抗原 1 (EBNA1)及 EB 毒溶裂誘導，使得細胞內 ROS大量增加，活化凋亡蛋白質 caspase-3 及 PARP 的裂解，進而誘發 B95-8 細胞的凋亡。以前文獻報導誘發 EB 病毒再活化因子除了丁酸鈉，還有生理 狀況, 如 IgG 或存活環境惡劣(0.1%FBS)等也會誘發 EB 病毒的再活化而導致腫瘤的發 生，而這些因子都能活化極早期基因 BRLF1 及 BZLF1 表現。而在 EB 病毒相關的腫瘤 會表現出不同類型的潛伏期蛋白質，潛伏期第一型 (Latency I)表現 EBNA1，在巴克氏淋巴瘤(BL)病人可以偵測到；潛伏期第二型 (Latency II) 表現 EBNA1、LMP1 及 LMP2 ，在鼻咽癌(NPC)、Hodgkin disease、T cell lymphoma 病人可以偵測到；潛伏期第三型 (Latency III) 表現 EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, EBNA-LP, LMP1, LMP2A, LMP2B 在免疫缺損相關的 B 淋巴瘤病人可以偵測到。基於先前研究結果我們認為虎杖分離物會影響不同 EB 病毒誘導劑及不同類型潛伏期腫瘤細胞，所以本計畫分三部份：第一部份、以細胞學實驗探討虎杖根分離物對不同溶裂循環誘導劑的影響。第二部份、以細胞學實驗探討虎杖根分離物對不同潛伏期EB病毒腫瘤細胞的影響。 第三部分，以動物模試探討虎杖分離物抗EB病毒及抗EB病毒腫瘤活性。我們將以半製備逆相式高效液相色層分析法將抗EB病毒及抗腫瘤有效成分分離純化。以流式細胞儀法及 real-time PCR 分析虎杖分離物對 EB 病毒溶裂蛋白質、ROS、EB病毒 DNA 及 mRNA 表現；以西方墨點法偵測EB病毒溶裂蛋白質、發炎因子及凋亡相關蛋白質的表現；以細胞毒性實驗及細胞核型態學觀察探討虎杖分離物抑制腫瘤細胞生長的影響，進 而以流式細胞儀定量凋亡的細胞數。並且以免疫螢光法分析虎杖分離物是否抑制腫瘤細胞內 NF-B 的表現。這些研究將有助於更了解虎杖根抗病毒及抗腫瘤功效。
From our recent studies demonstrated that ethanolic extract (PcE), ethyl acetate subfractions F1 and F3 from Polygonum cuspidatum root, resveratrol, emodin and piceid inhibit sodium butyrate-induced transcription of Epstein-Barr virus (EBV) immediate-early genes, including BRLF1 and BZLF1, and caused the expression of EBV lytic proteins decreasing, including Rta, Zta and EA-D. These inhibitions lead into suppress EBV DNA replication and virion production. Moreover, ethanolic extract, ethyl acetate subfractions F2a and F3 from Polygonum cuspidatum root and emodin induce apoptosis of EBV latency III tumor cells, B95-8 cells. PcE and ethyl acetate subfractions F3 promote B95-8 cells apoptosis due to its ability to inhibit the expression of latent membrane protein 1 (LMP1) and deplete NF-κB in the nucleus. The ethyl acetate subfraction F2a affects the expression of LMP1, EBV nuclear antigen 1 (EBNA1) and EBV lytic induction, induces high level of ROS production which lead to an increased caspase 3 activity and cleavage of PARP to trigger apoptosis of B95-8 cells. Previous literatures had reported that physiologic condition, IgG and 0.1% FBS also can induce EBV reactivation except sodium butyrate, enhance profoundly an in tumorigenesis. All these factors can activate the expression of EBV immediate-early genes, including BRLF1 and BZLF1. In all EBV-associated malignancies that the virus establish a latent infection characterized by different EBV gene expression profiles, EBNA1 expressed in latency I mainly found in EBV-positive Burkitt’s lymphoma, latency II expressed EBNA1, LMP1 and LMP2 mostly related to nasopharyngeal carcinoma, latency III defined by the expression of EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, EBNA-LP, LMP1, LMP2A, LMP2B presented in most immunodeficiency-related B cell lymphoma. Based on the pilot studies, I propose that the ethyl acetate subfractions of Polygonum cuspidatum root may affect IgG or 0.1% FBS induced EBV reactivation and inhibit proliferation and survival of EBV-positive tumor cells depending on viral latency program. Therefore, in this proposed study, I will to perform three parts: Part I and Part II, Using cell culture model to explore the effect of ethyl acetate subfractions of Polygonum cuspidatum on a variety of EBV reactivation inducer and EBV-positive tumor cells depending on viral latency programs. Part III, Using animal model to investigate the effect on the anti-EBV activity and therapeutic efficacy for treating EBV-positive tumors. Semi-preparative reverse phase high performance liquid chromatography will be used to isolate the antiviral and anti-tumor subfractions. To quantitate the expression of EBV lytic proteins and ROS and to determine the EBV DNA and mRNA expression using flow cytometric analysis and real-time PCR. Immunoblotting assay will used to determine the expression of EBV lytic proteins, inflammation factors and apoptosis-related proteins. I will perform the cytotoxicity assay and nuclei morphology observation used to analyze whether ethyl acetate subfractions have the anti-proliferative activity on EBV-positive tumor cells. Indirect immunofluorescence was performing to investigate the NF-B expression. The study is important for elucidating the antiviral and antitumor efficacy of Polygonum cuspidatum root. |
