- 學位論文
中草藥對EB病毒感染週期的影響
The Effects of Chinese Herbs on the Infectious Cycle of Epstein-Barr Virus
摘要
EB病毒 (Epstein-Barr virus, EBV)是廣泛感染人類的疱疹病毒，它主要感染淋巴細胞和上皮細胞。病毒在感染B淋巴細胞時會造成潛伏感染並導致細胞的不死化 (immortalization)。目前全球大約90%的成年人都曾經感染過EB病毒。在感染的生活使中，EB病毒會表現許多病毒基因，其分別負責調控著病毒感染進入潛伏期 (latent stage) 或者溶裂期 (lytic stage)。在臨床研究上，EB病毒與許多疾病及惡性腫瘤有關，包括感染性單核球增多症 (infectious mononucleosis) 巴克氏淋巴瘤 (Burkitt’s lymphoma) 和鼻咽癌 (nasopharyngeal carcinoma) 等，但至今沒有好的治療藥劑。所以尋找有效的治療藥劑是本論文研究的方向。我們依據前人的研究以及中草藥的生物特性，選出298種中草藥的粗萃取物以及純化物進行研究。首先利用流式細胞儀技術 (flow cytometry) 偵測這些中草藥對病毒複製的影響。結果顯示，其中有24種明顯影響EB病毒的複製。隨後再利用即時聚合酶連鎖反應 (Real-Time Polymerase Chain Reaction, Real-Time PCR) 技術做進一步的確認，發現在兩種技術偵測下，共有7種確實會抑制EB病毒複製，2種會促進病毒複製，且隨著濃度改變而改變病毒複製能力。在另一方面，我們利用反轉錄聚合酶連鎖反應 (Reverse transcription polymerase chain reaction, RT-PCR) 技術進一步探討24種中草藥對病毒感染週期的影響。結果發現大葉桃花心木正丁醇萃取物會抑制EB病毒潛伏基因LMP1和極早期基因BZLF1的表現，但不會影響晚期基因BLLF1的表現。之後發現有10種中草藥萃取物會抑制LMP1的表現，但不會影響BZLF1以及BLLF1。其中又以升麻、4-methoxychalcone以及oxopurpureine最為明顯。繼續分析又發現有3種中草藥萃取物會活化LMP1的表現，但不會影響BZLF1和BLLF1的表現，其中以淡豆豉及石胡荽最為明顯。最後還有3種中草藥萃取物一方面會抑制LMP1的表現，而另一方面又會活化BZLF1的表現，但不會影響BLLF1的表現。其中以相思樹莖部及補骨脂的甲醇萃取物最為明顯。這結果表示這些中草藥可能在EB病毒複製及感染週期中扮演重要的角色，因此在未來的研究上，我們將從這些有效中草藥萃取物著手，找尋影響EB病毒的主要成份，並進一步探討其機制，以提高EB病毒相關疾病的治療。
並列摘要
Epstein-Barr Virus (EBV) is a human herpesvirus, which infects both lymphocytes and squamous epithelial cells. This virus has an instinct, which can immortalize the primary B lymphocytes into lymphoblastoid cell lines (LCLs) in vitro. EBV encoded several genes (latent and lytic genes), which are involved in the regulation of the entry of latent or lytic stage in the infectious cycle. At present, there are greater than 90% adult poputation with EBV infection in the word. EBV is closely associated with the clinical diseases and development of malignancies including infectious mononucleosis, Burkitt’s lymphoma and nasopharyngeal carcinoma. However there is no approved or effective medicine for EBV treatment so far. Therefore the main goal of our thesis is to search for the new and effective medicines for treatment of EBV associated disearses. Previously, the accumulating evidences have demonstrated that the active components from Chinese herbs function as biological activity. In this study, we first select 298 Chinese herbs comprising crude extracts and pure compounds to test whether these Chinese herbs affect EBV replication under flow cytometry analysis. These results indicate that 24 Chinese herbs show more significant. Then we further confirm the function of these 24 Chinese herbs in EBV replication under Real-Time PCR analysis. The results show that 7 Chinese herbs decrease EBV replication and 2 Chinese herbs increase EBV replication under the analysis of these two methods. In the same time, these 9 replication affecting Chinese herbs are dose-dependent. On the other hand, we use RT-PCR to examine whether these 24 Chinese herbs affect EBV infectious cycle. These results show that Swietenia macropnylla makedly reduces the expression of the latent gene, LMP1 and immediate early gene, BZLF1, but has no effect on late gene, BLLF1. In addition, Cimicifuga foetida , 4-methoxychalcone and oxopurpureine inhibit the LMP1 expression, but have no effect on the expression of BZLF1 and BLLF1. Glycine max and Centipeda minima only increase the expression of the LMP1, but has no effect on other gene expression. Finally we find out two herbal medicines, Acacia confusa and Psoralea corylifolia , which decrease the LMP1 expression. On the other hand, these two herbal medicines also increase the expression of the BZLF1, but have no effect on BLLF1. Taken together, the above data indicate that these crude extracts or pure compunds may play an important role in regulating the EBV replication and infectious cycle. In the future, we will further search for the active components of these effective Chinese herbs on EBV infection. These will be potential for use in the treatment of EBV associated diseases.