臺灣博碩士論文加值系統

日本腦炎病毒(Japanese encephalitis virus, JEV)為正向單股RNA病毒，屬於黃病毒科病毒。日本腦炎病毒的感染大部分為無症狀感染，少部分輕微病例會產生頭痛、發燒、昏迷、痙攣、無菌性腦膜炎等症狀或死亡。日本腦炎的治療仍以支持療法為主，在目前尚未有有效的治療方法。石榴皮(Pericarpium granati, PG)煎劑是傳統中藥，常用於抗病毒，如在雞胚胎實驗中對流感病毒具有抑制作用，在細胞培養實驗也同樣具有抗疱疹病毒的能力。本研究探討石榴皮水萃物與石榴皮主成分Punicalagin抑制日本腦炎病毒感染細胞的功效及機轉。首先以MTT 試驗測得石榴皮水萃物對BHK-21細胞之半毒殺濃度(CC50)為53μg/mL，而對TE-671細胞之CC50為219.7μg/mL，在Punicalagin對BHK-21細胞的CC50為14μM，以及對TE-671細胞的CC50為42μM。石榴皮水萃物與Punicalagin具有劑量依賴模式抑制日本腦炎病毒感染BHK-21與TE671細胞株的細胞病變現象(Cytopathic Effect Assay ,CPE assay)。分別以先給藥一小時、先感染一小時或同時給藥與感染的條件下，以病毒蝕斑試驗(Plaque assay)計算抑制病毒斑點百分之五十的藥物濃度，發現石榴皮水萃物在先給藥再感染模式之藥物抑制日本腦炎病毒其病毒半抑制藥物濃度(IC50)為4.4μg/mL，同時給藥並感染模式之IC50為5.2μg/mL，而先感染後給藥之抑制日本腦炎病毒IC50則為74.6μg/mL；同樣地Punicalagin先給藥後感染的IC50為1.17μM，同時給藥感染模式的IC50為3.27 μM，先感染後給藥模式的IC50為9.18μM。由此結果指出，石榴皮水萃物與Punicalagin對日本腦炎病毒具有預防病毒感染細胞的能力。而在免疫路徑驗證的實驗中，發現NF-κB 蛋白會隨著石榴皮水萃物與指標成分Punicalagin濃度增加而增加其表現量，因此證明石榴皮水萃物與指標成分Punicalagin以會誘導細胞產生免疫發炎因子NF-κB的這條訊息路徑產生免疫反應抵抗病毒的感染。

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus with positive-sense single-stranded RNA genome. JEV infection is asymptomatic, only few cases have headache, fever, encephalitis, coma and even death. Treatment for JE is supportive and still lack of efficacy. Water extrats of Pericarpium granati (PG) is an traditional herbal therapeutic with antiviral activities. It was shown to inhibit influenza A virus replication in chicken embryos and herpes simplex viruses in cell culture. In this study, we intend to investigate the anti-JEV activities and functional mechanism of PG water extract and the related compound punicalagin (PN). CC50 values (50% cytotoxicity concentration) of PG water extract by MTT test were 53 μg/mL for BHK-21 cells and 219.7 μg/mL for TE-671 cells, while CC50 values of PN were14 μM for BHK-21 cells and 42.7 μM for TE-671 cells. PG water extract and PN showed a concentration-dependently inhibitory effect on JEV-infected cytophatic effect and virus production in BHK-21 and TE-671 cells. The plaque reduction abilities of PG water extract and PN were studied by three different procedures including addition at 1 hour before virus inoculation (pre-treatment), addition and virus inoculation at the same time (simultaneous treatment), and addition at 1 hour after virus inoculation (post-treament). IC50 values (50% inhibition concentration) of PG water extract were 4.4 μg/mL by pre-treatment, 5.2 μg/mL by simutaneous treatment and 74.6 μg/mL by post-treament. IC50 values (50% inhibition concentration) of PN were 3.27 μM by pre-treatment, 1.17 μM by simutaneous treatment and 9.18 μM by post-treament. In vivo signaling pathway assay indicated PG water extract and PN inducing activation of the NF-κB signaling pathway.

誌謝 I
中文摘要 III
英文摘要 V
目錄 VI
圖目錄 VIII
表目錄 XI
第一章、前言 1
第一節、研究背景 1
一、日本腦炎 1
二、日本腦炎病毒 2
三、日本腦炎流行病學 2
四、日本腦炎病毒之基因與複製機轉 3
五、日本腦炎病理特性 5
六、日本腦炎現今治療方式 6
七、石榴 7
八、石榴化學成分與藥理作用 7
九、石榴皮藥理作用 8
第二節、研究目的 10
一、研究動機 10
二、研究目的 10
第二章、研究方法 11
第一節、研究設計 11
第二節、研究材料 13
第三節、實驗方法 16
一、中藥製備 16
二、冷凍細胞之解凍活化(Thawing frozen cells) 16
三、細胞冷凍保存( Cryopresezvation ) 17
四、細胞記數法( Cell count ) 17
五、細胞培養( Cell culture ) 18
六、細胞存活試驗( MTT cell viability assay ) 18
七、病毒增幅放大( Virus amplification ) 20
八、病毒感染細胞之細胞病變實驗(Cytopathic effect assay ) 20
九、病毒蝕斑試驗( Plaque assay ) 21
十、細胞RNA之抽取(Cell RNA purification) 21
十一、逆轉錄酶-聚合酶連鎖反應( Reverse transcription PCR ) 22
十二、即時定量聚合酶連鎖反應( Real-Time PCR ) 23
十三、同源冷光表現系統( Cis-acting reporter assay system ) 24
第三章、實驗結果 26
一、利用Plaque assay 定量日本腦炎T1P1病毒株的病毒量。 26
二、定量石榴皮水萃物對BHK-21與TE-671細胞半致死濃度CC50。 26
三、定量石榴皮水萃物對BHK-21與TE-671細胞CPE reduction assay觀察藥物抑制病毒感染BHK-21細胞之現象。 27
四、定量石榴皮水萃物對BHK-21與TE-671病毒半抑制濃度IC50。 27
五、石榴皮水萃物與指標成分Punicalagin誘導細胞免疫反應路徑分析。 28
六、石榴皮水萃物與指標成分Punicalagin訊息路徑確認。 29
第四章、討論 31
第五章、結論與建議 34
第一節、結論與建議 34
參考文獻 37
附錄 41
圖 41
表 84

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