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研究生:王秋雅
研究生(外文):Qiu-ya Wang
論文名稱:中草藥萃取物對於腸病毒71型感染人類肌肉細胞效用之分析
論文名稱(外文):The effects of Chinese medical herbs treatment on Enterovirus 71 infection in rhabdomyosarcoma cells
指導教授:林雅菁林雅菁引用關係
指導教授(外文):Ya-Ching Lin
學位類別:碩士
校院名稱:輔英科技大學
系所名稱:生物技術系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:68
中文關鍵詞:腸病毒71型中草藥萃取物
外文關鍵詞:water extracts of chinese medical herbsEnterovirus 71
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本研究利用藿香、白芷、半枝蓮葉、半枝蓮根、丹參、栝樓以及胡蘆巴等7種中草藥萃取物,以人類橫紋肌瘤細胞株(Rhadomysarcoma cell line)為測試系統,檢測這些萃取物對於腸病毒感染細胞作用的影響,藉由不同條件下處理細胞與病毒、萃取物處理次數的不同和不同感染複數(MOI , multiplicity of infection)的效用下觀察細胞存活率。我們實驗結果顯示各種中草藥水溶液萃取物經不同方式處理細胞或病毒,會產生不同的腸病毒毒殺細胞的效用。使用藿香水溶液萃取物與腸病毒混和,能產生延緩在後感染過程中腸病毒釋放的效用,但卻不能有效抑制腸病毒的生長。在後感染過程中,病毒培養基持續性加藥(每隔12小時)可將細胞存活率由29%提升至61%。分析細胞內外病毒力價得知,每隔12小時加藥將會抑制細胞內病毒的複製,同時也抑制腸病毒的釋放至細胞外, 進而達到降低細胞毒殺性的效果。
In this study, seven water extracts of chinese medical herbs were used for evaluating the effects of their treatment on the infection of enterovirus 71(EV71) on rhabdomyosarcoma cells (RD). The virus with different concentration or RD cells were pre-treated with water extract before or after infection, combined with interval addition of extract into the medium during the period of post-infection. Our results showed that the treatment of different medical herb extracts on cells or enterovirus 71 displayed different effects. By the using of Agastache rugosa extract with EV71 prior to the infection to RD cells, a significant delay on cell death was observed after the treatment, where the virus release into medium was delayed; however, the virus production in the cells was not inhibited. By the addition of the extract into medium at intervals of 12h, the virus production in the cells was inhibited and the virus release into medium was delayed, leading to the delay on cell death caused by the EV71 infection.
目 錄

誌謝 ------------------------------------- i
中文摘要 ------------------------------------ ii
英文摘要 -------------------------------------------- iii
目錄 ---------------------------------------- iv
表目錄 ------------------------------- vi
圖目錄 ------------------------------------- vii
縮寫表 ---------------------------------- x
第一章 緒論--------------------------------- 1
第二章 文獻探討---------------------------- 2
第一節 腸病毒71型------------------------- 2
第二節 中草藥在抗腸病毒71型之研究近況-------- 5
第三節 科學中草藥的發展------------------ 7
ㄧ 藿香------------------------------------- 7
二 白芷------------------------------------- 9
三 胡蘆巴------------------------------------ 11
四 栝樓---------------------------------------- 12
五 丹參----------------------------------- 13
六 半枝蓮------------------------------ 14
第三章 研究方法----------------------------- 16
第一節 藥品與材料------------------------- 16
第二節 細胞培養-------------------------- 17
第三節 腸病毒的培養-------------------------- 18
第四節 中草藥製備--------------------- 18
第五節 細胞存活率檢測(本實驗分別使用MTT與LDH來檢測細胞的存活率)---------------------------------- 19
一 MTT檢測------------------------------ 19
二 LDH檢測----------------------------------- 19
第六節 病毒力價(TCID50)--------------------- 20
第七節 實驗設計---------------------------- 21
第八節 實驗流程的目的----------------------- 22
第四章 結果與討論----------------------------- 23
第一節 中草藥萃取物對於細胞毒性分析-------- 23
第二節 中草藥水溶液萃取物抗腸病毒71型感染細胞的效用- 24
第三節 藿香水溶液萃取物抗腸病毒71型感染細胞的效用 29
一 萃取物處理次數之影響-------- 29
二 不同MOI感染的效用--------- 31
第五章 結論與建議------------------------ 33
參考文獻 ---------------------- 34
圖 --------------------------------------- 44
附錄 ---------------------------------------- 68
附錄一 --------------------------------- 68





































表 目 錄

表一 腸病毒分類--------------------------- 3
表二 中草藥水溶液萃取物經各種方法處理腸病毒71型及細胞-
24























圖 目 錄


圖一
Ro 09-0179和Ro 09-0298結構式---------- 8
圖二
藿香水溶液於各種濃度下對細胞毒性之分析---- 44
圖三
白芷莖水溶液於各種濃度下對細胞毒性之分析 45
圖四
丹參水溶液於各種濃度下對細胞毒性之分析- 46
圖五
半枝蓮根水溶液於各種濃度下對細胞毒性之分析- 47
圖六
半枝蓮葉水溶液於各種濃度下對細胞毒性之分析-- 48
圖七
胡蘆巴水溶液於各種濃度下對細胞毒性之分析--- 49
圖八
栝樓水溶液於各種濃度下對細胞毒性之分析--- 50
圖九
8mg藿香水溶液萃取物使用不同條件處理細胞與病毒後,細胞在後感染過程中的存活率-----51
圖十
8mg半枝蓮根水溶液萃取物使用不同條件處理細胞與病毒後,細胞在後感染過程中的存活率------------------52
圖十一 8mg半枝蓮葉水溶液萃取物使用不同條件處理細胞與病毒後,細胞在後感染過程中的存活率------------------53
圖十二 6mg白芷莖水溶液萃取物使用不同條件處理細胞與病毒後,細胞在後感染過程中的存活率---------------------54
圖十三 160mg胡蘆巴水溶液萃取物使用不同條件處理細胞與病毒後,細胞在後感染過程中的存活率-----------------55
圖十四 10mg栝樓水溶液萃取物使用不同條件處理細胞與病毒後,細胞在後感染過程中的存活率---------------------56
圖十五 6mg丹參水溶液萃取物使用不同條件處理細胞與病毒後,細胞在後感染過程中的存活率------------------------57
圖十六 8mg藿香水溶液與腸病毒混合三小時後再感染細胞ㄧ小時(吸附作用),再用不同培養條件進行病毒培養,其對細胞存活率之影響-------------------------------------58
圖十七 在後感染過程中,進行藿香萃取物的加入對於病毒與細胞的效用,藿香與腸病毒混和3小時後,再將此混和液感染細胞後,於後感染過程中進行不同處理,於第三十六小時的細胞圖---------------------------59
圖十八 8mg藿香水溶液與腸病毒混合三小時後再感染細胞ㄧ小時(吸附作用),再用不同培養條件進行病毒培養,其對細胞內病毒力價之影響--------------------------------60
圖十九 8mg藿香水溶液與腸病毒混合三小時後再感染細胞ㄧ小時(吸附作用),再用不同培養條件進行病毒培養,其對細胞外病毒力價之影響--------------------------------61

圖二十
8mg藿香水溶物萃取物與不同的濃度腸病毒71型(MOI= 0.1)混和作用1、3或5小時後,再加入細胞中感染一小時,於後感染過程中使用病毒培養基進行培養,其細胞存活率之情形-----------------62
圖二十一 8mg藿香水溶物萃取物與不同的濃度腸病毒71型(MOI=0.1)混和作用1、3或5小時後,再加入細胞中感染一小時,於後感染過程中使用病毒培養基進行培養,其細胞內病毒生長之情形--------------------63
圖二十二 8mg藿香水溶物萃取物與不同的濃度腸病毒71型(MOI=0.1)混和作用1、3或5小時後,再加入細胞中感染一小時,於後感染過程中使用病毒培養基進行培養,其細胞外病毒力價之情形----------------64
圖二十三 8mg藿香水溶物萃取物與不同的濃度腸病毒71型(MOI= 0.01)混和作用3小時後,再加入細胞中感染一小時,於後感染過程中使用病毒培養基進行培養,其細胞存活率之情形-------------65
圖二十四 8mg藿香水溶物萃取物與不同的濃度腸病毒71型(MOI=0.01)混和作用1、3或5小時後,再加入細胞中感染一小時,於後感染過程中使用病毒培養基進行培養,其細胞內病毒力價之情形------------------------66
圖二十五 8mg藿香水溶物萃取物與不同的濃度腸病毒71型(MOI=0.1)混和作用1、3或5小時後,再加入細胞中感染一小時,於後感染過程中使用病毒培養基進行培養,其細胞外病毒力價之情形---------------------------67
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