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研究生:謝繼璋
研究生(外文):Chi-Chang Hsieh
論文名稱:大王椰子萃取物抗單純疱疹病毒第一型活性之分析
論文名稱(外文):Analysis of Anti-Herpes Simplex Virus Type 1 Activity from Roystonea regia Extract
指導教授:郭育綺郭育綺引用關係
指導教授(外文):Yuh-Chi Kuo
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:83
中文關鍵詞:單純疱疹病毒第一型大王椰子
外文關鍵詞:Herpes simplex virus type 1
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本研究藉由找尋中草藥來對抗單純疱疹病毒第一型。利用病毒斑減少檢測 ( Plaque reduction assay ) 鑑定出大王椰子 ( Roystonea regia ) 萃取物在不毒殺宿主細胞情況下,可有效降低 HSV-1 病毒斑形成,其 IC50 為46.94 μg/ml。研究目標為以下兩項:(1) 分析大王椰子萃取物在動物體內抗病毒活性;(2) 分析大王椰子萃取物可能之作用機轉。研究結果顯示:(A) 將病毒感染BALB/c 小鼠之皮膚,並分別餵食低劑量 (140 mg/kg) 與高劑量 (210 mg/kg) 大王椰子萃取物,對照組餵食 Corn oil,每日兩次並持續餵食一星期。結果發現疱疹病徵可被高劑量大王椰子萃取物改善;(B) 抽取BALB/c小鼠皮膚DNA,以Real-Time PCR方法偵測皮膚中HSV-1 DNA表現量,發現餵食高劑量大王椰子萃取物皆可以降低HSV-1 DNA在BALB/c小鼠皮膚中表現量;(C) 抽取BALB/c小鼠血液分離血清,以試劑偵測其中肝功能Asparate aminotransferase ( AST ) 與Alanine aminotransferase ( ALT ) 及腎功能 Blood urea nitrogen ( BUN ) 與Creatinine ( CRE ) 指數,發現大王椰子萃取物無論是高劑量或低劑量情況下,對於小鼠並不具有肝毒性與腎毒性;(D) 利用 HSV-1 感染 Vero 細胞後不同時間加入大王椰子萃取物、Vero 細胞前處理大王椰子萃取物再進行病毒感染或病毒與天然物同時加入細胞等實驗,再檢測病毒斑形成情形。發現大王椰子萃取物在前處理或與病毒同時加入情況下,具有最高之抑制活性。因此推論大王椰子萃取物抑制 HSV-1 複製主要機轉可能與影響病毒之吸附 ( Adsorption ) 過程有關;(E) 利用4度環境下,將細胞前處理以天然物不同時間再洗掉並接著感染病毒,藉由病毒斑檢測的結果,顯示大王椰子萃取物並無抑制病毒複製的能力,推測天然物並非鍵結於細胞上之病毒Receptor而抑制了HSV-1吸附過程;(F) 以大王椰子萃取物於37度環境下處理 HSV-1一小時,利用病毒斑減少法測試病毒活性及Real-Time PCR偵測病毒基因表現,發現大王椰子萃取物可能透過破壞或改變病毒結構而阻止病毒進入細胞,進而達到抗病毒之活性。(G) EMSA 實驗證實大王椰子可以有效抑制病毒活性且不會促使細胞核內NF-kB活化。最後希望藉由此研究成果,可成為抗病毒新藥開發之可能性。
Herpes simplex virus type 1 (HSV-1) belongs to Herpesviridae and causes cold sores in human. Although nucleotide analogs have been used as anti-viral drugs, there are drug-resistant HSV-1 strains found in clinical. Therefore, it’s important to identify new medicines against HSV-1 infection. In behalf to find new anti-HSV-1 compund, this study discocery extracts from Roystonea regia significantly blocked HSV-1. In the present study, the extracts from Roystonea regia significantly blocked HSV-1 replication in Vero cells without apparent cytotoxicity. Its IC50 is 46.94 μg/ml. To elucidate R. regia anti-HSV-1 activity in vivo, BALB/c mice were injected subcutaneous with HSV-1 ( 4×106 PFU ), treated orally with acyclovir ( 60 mg/Kg ) or R. regia ( 140 and 210 mg/Kg ) for 7 days, and inspected daily for signs of disease. Data from the scoring system indicated that animals infected with HSV-1, developed progressive zoster lesions. In marked contrast to the results with control mice, treatment with acyclovir or R. regia resulted in a sustained protective effect. No toxic effect of R. regia on liver and kidney functions was apparent. In order to realize what point of R. regia inhibits HSV-1 activity, I used time of addition experiment to figure out its mechanism. When pre-treat or treat R. regia at the same time, R. regia can significantly inhibit HSV-1 activity. So, it suggested that R. regia may have an effect on HSV-1 replication due to block the adsorption of HSV-1. To confirm above suggestion, first I pre-treat Vero cells and R. regia at 4C and then add 100 PFU of HSV-1 into cell. In plaque reduction assay result indicated that there was no difference between control and experiment groups, R. regia may not block the receptor of cell to reduce HSV-1 activity. In another way, I pre-treat HSV-1 and R. regia at 37C 1hr and then add 100 PFU of HSV-1 into cell. Also in plaque reduction assay and Real-time PCR results indicated that there were significant differences between DMSO and R. regia groups even there was a dose dependant on plaques and expression ratio. Results indicated that R. regia may disrupt or change conformation of HSV-1 and account for inhibition of HSV-1 replication in vitro.EMSA results also improves that R.r extracts can decrase HSV-1 activity and inhibit NFB activation.These results indicated that R. regia was a potent inhibitor of the in vitro and in vivo replication of HSV-1. Moreover, this study not only demonstrates Chinese herbs are potential therapeutic drugs for the viral infection but also support a model for future protocol design in preclinical studies.
縮簡表
中文摘要
英文摘要
壹、前言
貳、材料與方法
參、結果
肆、討論
伍、參考文獻
陸、表
柒、圖
捌、附錄
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