臺灣博碩士論文加值系統

腸病毒71型 (Enterovirus 71, EV71)屬於小RNA病毒科中腸病毒A型，可經由糞口傳播入侵人體，最常見的症狀為手足口症，對於年幼患者更可能產生嚴重的中樞神經性併發症，而導致差的預後甚至死亡，目前沒有有效疫苗和藥物去預防及治療。薑黃素為薑黃植物根莖中的主要成分，其相關研究指出薑黃素具有抵抗細菌、寄生蟲以及病毒的感染。研究實驗中利用薑黃素處理大腸癌細胞HT29細胞，再進行EV71病毒感染並觀察病毒是否受到薑黃素的影響，在實驗結果中發現經薑黃素處理之HT29細胞會減低EV71的複製能力。為了進一步了解薑黃素影響EV71感染的機制，我們分別探討與EV71感染相關之細胞訊號傳遞蛋白質p38、Erk1/2，結果顯示細胞內的p38和Erk1/2改變，並沒有影響EV71的感染力。有文獻指出薑黃素具有抗氧化的功能，實驗結果中顯示腸病毒感染在HT29細胞早期不會引發ROS上升。而已經有些酵素能影響病毒的複製，當中包括PKC，而PKC對於病毒的影響主要發生在早期，因此我們偵測薑黃素對PKC的影響，發現PKC的磷酸化會因薑黃素而被抑制，同時EV71的蛋白質表現量也減少，利用PKC的抑制劑rottlerin去處理細胞，同樣能抑制病毒的感染力，因此我們認為薑黃素是藉由抑制PKC的磷酸化從而影響到EV71的感染力。

Enterovirus 71 (EV71) belongs to genus Enterovirus of the family Picornaviridae. EV71 is transmitted through the fecal-oral route. The most common symptom of EV71 infection is hand-foot-mouth disease. This virus may cause severe neurological complications in young children. There is no effective agent or vaccine against EV71 infection. Curcumin is the main component of Curcuma longa L. Curcumin has the antimicrobial activity against bacteria, fungi and virus. We investigated the effects of curcumin in EV71 infected HT29 cells. The results showed that the expression of viral RNA, protein and viral particle were reduced in curcumin-treated HT29 cells. In order to assess the mechanism of curcumin, we examined the expression of p38 and Erk. An increased phosphorylation of p38 and Erk in the curcumin-treated cell was demonstrated. Relevant studies showed that EV71 can modulate the ROS production in cells in favor to viral replication. Our data revealed that EV71 increased the ROS in early phase and curcumin does not have significant antioxidant properties. PKCis associated with virus infection. We examined the phospho-PKC on curcumin treated HT29 cells, the result showed curcumin inhibited the phosphorylation of PKC. The PKC inhibitor significantly inhibited the phosphorylation of PKC and reduced the EV71 replication. Therefore, curcumin may reduce the EV71 replication through inhibition of the PKC phosphorylation.

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致謝........................................................................................................... iii
中文摘要................................................................................................... iv
Abstract......................................................................................................v
目錄........................................................................................................... vi
圖目錄.........................................................................................................x
表目錄...................................................................................................... xii
一、 前言...............................................................................................1
1-1、腸病毒 71 型流行病學..............................................................1
1-2、腸病毒的分類 ............................................................................1
1-3、腸病毒 71 型的構造及基因結構..............................................2
1-4、腸病毒 71 型的生活史..............................................................3
1-5、薑黃素的簡介 ............................................................................4
1-6、薑黃素的抗病毒活性................................................................4
1-7、p38 的信息傳遞路徑.................................................................5
1-8、Erk 的信息傳遞路徑.................................................................5
1-9、protein kinase C  的信息傳遞路徑........................................6
二、 實驗目的.......................................................................................7
三、 實驗材料與方法...........................................................................8
3-1、細胞培養 (subculture) .............................................................8
3-1-1、RD 培養 ...........................................................................8
3-1-2、HT29 培養 .......................................................................9
3-2、實驗所用之病毒株....................................................................9
3-2-1、EV71 4643 .......................................................................9
3-3、病毒放大 (Amplification)......................................................10
3-4、plaque assay.............................................................................11
3-6、病毒感染 (Infection)..............................................................12
3-7、抽取細胞內的 RNA.................................................................12
3-8、RT-PCR....................................................................................13
3-9、DNA 電泳................................................................................14
3-10、real-time PCR(Quantitative PCR, qPCR) .........................15
3-11、西方墨點法 (Western Blot) .................................................15
3-12、螢光免疫染色 (immunofluorescence assay, IFA) .............17
3-13、DCFDA 染色 .........................................................................18
四、 實驗結果.....................................................................................20
4-1、探討薑黃素影響腸病毒 71 型感染力的分子機制部份...............20
4-1-1、EV71 感染 HT29 細胞後有明顯的病毒蛋白質 3D 表現 .20
4-1-2、EV71 對 HT29 細胞具有高感染率 ...................................20
4-1-3、濃度高的薑黃素處理 HT29 細胞會使細胞死亡...............21
4-1-4、EV71 感染經薑黃素處理過的 HT29 細胞後其病毒 RNA
和病毒顆粒的產生明顯減少 ...........................................................22
4-1-5、EV71 感染 HT29 細胞後會誘發信息傳遞蛋白質 p38 和
Erk 蛋白質活化................................................................................22
4-1-6、薑黃素處理 HT29 細胞後增加細胞內 p38 及 Erk 活化並
減少病毒蛋白質的表現....................................................................23
4-1-7、減少細胞信息傳遞蛋白質 p38 和 Erk 的活化並不影響病
毒蛋白質的表現................................................................................24
4-1-8、薑黃素具有少量的抗氧化能力...........................................25
4-1-9、薑黃素處理 HT29 細胞後抑制細胞內 PKC 活化並減少
病毒蛋白質的表現............................................................................26
4-1-10、Rottlerin 抑制病毒感染細胞所造成的細胞病變現象....26
4-1-11、Rottlerin 抑制病毒顆粒的表現量。 ................................27
4-1-12、Rottlerin 透過抑制細胞內 PKC 的活化從而減少病毒蛋
白質的表現。....................................................................................28
五、 實驗討論.....................................................................................30
六、 參考文獻.....................................................................................34
七、 附圖.............................................................................................38
八、 附表.............................................................................................52

目錄
圖 一、各種中草藥對抗 EV71 感染之機制分類圖。...........38
圖 二、薑黃素對抗病毒感染之機制。 ..................................39
圖 三、EV71 感染 HT29 後細胞內病毒蛋白質 3D 以及病毒
顆粒的表現量。.................................................................40
圖 四、EV71 感染 HT29 後的病毒蛋白質 3D 在細胞內的表
現情形。.............................................................................41
圖 五、薑黃素處理 HT29 後的細胞存活情況。...................42
圖 六、EV71 感染經薑黃素處理的 HT29 細胞後其病毒複製
情形。.................................................................................43
圖 七、EV71 感染 HT29 後細胞內的信息傳遞蛋白質表現情
形。.....................................................................................44
圖 八、EV71 感染經薑黃素處理的 HT29 細胞其病毒蛋白質
和 RNA 以及細胞信息傳遞蛋白質 p38 和 Erk 的表現情
形。.....................................................................................45
圖 九、利用抑制劑抑制信息傳遞蛋白質後對 EV71 感染沒
有影響。.............................................................................46
圖 十、薑黃素具有少量的抗氧化能力。 ..............................47
圖 十一、薑黃素處理 HT29 細胞後抑制細胞內 PKC 活化
並減少病毒蛋白質的表現。.............................................48
圖 十二、Rottlerin 有效抑制細胞死亡。..............................49
圖 十三、Rottlerin 抑制病毒顆粒的表現量。......................50
圖 十四、Rottlerin 透過抑制細胞內 PKC 的活化從而減少
病毒蛋白質的表現。.........................................................51

表目錄
表格 一、PCR primer 列表 ....................................................52

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