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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 張鑫 | |
dc.contributor.author | Yen-Chang Chen | en |
dc.contributor.author | 陳彥璋 | zh_TW |
dc.date.accessioned | 2021-05-20T20:33:26Z | - |
dc.date.available | 2010-09-11 | |
dc.date.available | 2021-05-20T20:33:26Z | - |
dc.date.copyright | 2008-09-11 | |
dc.date.issued | 2008 | |
dc.date.submitted | 2008-07-30 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/9648 | - |
dc.description.abstract | C型肝炎病毒 (Hepatitis C virus, HCV) 非結構性蛋白質NS4B為一鑲嵌於內質網之疏水性蛋白質,能改變C型肝炎病毒感染細胞內質網的構形並形成網狀膜(membranous web),而此網狀膜為病毒複製自身基因的位置;目前在病毒複製及細胞毒害方面,NS4B蛋白質的作用仍不清楚。先前本實驗室研究指出,當在癌化肝細胞Huh-7及腎細胞293表現NS4B蛋白質時,細胞中的Fas-activated serine/ threonine kinase (FASTK)的mRNA量有下降的趨勢。本實驗首先建立一穩定細胞株—HepG2-2-NS4B,此細胞株能經由doxycycline誘導表現NS4B蛋白質;藉由real time PCR觀察,此穩定細胞株經誘導表現NS4B蛋白質時,細胞中的FASTK的mRNA量相較於未誘導表現NS4B蛋白質之細胞減少五成。先前已知FASTK能調控細胞中Fas mRNA的選擇性剪接,使細胞轉譯出具相反生理意義之Fas isoforms,進而對於Fas活化產生不同程度之耐受度。因此在本實驗中,利用ELISA檢視細胞製造、分泌至細胞外之soluble Fas及細胞中所有Fas的量,結果發現誘導表現NS4B蛋白質的穩定細胞株和母細胞株相較時,NS4B穩定細胞株釋放至培養基的soluble Fas的量是母細胞株的2.5倍、而其釋放至培養基的soluble Fas佔所有Fas的比例則為母細胞株的1.4倍。當利用一能活化Fas 受器之anti-Fas抗體去誘導穩定細胞株進行細胞凋亡,再藉由trypan blue exclusion assay去監測細胞存活率則發現,穩定細胞株誘導表現NS4B蛋白質時,其細胞存活率為未受誘導之穩定細胞株的1.7倍;然而進一步檢視穩定細胞株受到anti-Fas抗體刺激後caspase 3活化狀況,發現接受anti-Fas抗體刺激後NS4B穩定細胞株之caspase 3活性降低到母細胞株的41%。推測soluble Fas量的上升及caspase 3活化程度的降低有直接關聯,而FASTK mRNA的下降也許參與其中;然而細胞存活率的上升可能是由於NS4B蛋白質藉由影響FASTK或其它細胞因子,進而調控細胞凋亡以外的訊息傳遞過程所造成,至於真正造成上述現象之機轉則待進一步實驗去釐清。 | zh_TW |
dc.description.abstract | Nonstructural protein 4B (NS4B) of hepatitis C virus (HCV) is a hydrophobic protein that integrates into endoplasmic reticulum (ER) and causes the formation of membranous web, a hypothetical site for viral replication in the cells. However, the roles of NS4B involved in virus life cycle and pathophysiologic effects remain elusive. Results from our previous studies indicate that NS4B plays a role in the down regulation of the expression of Fas-activated serine/threonine kinase (FASTK) mRNA. In this study, the stable cell line HepG2-2-NS4B that can express NS4B protein upon doxycycline induction was established. The level of FASTK mRNA declined to 51% in the doxycycline-induced, NS4B-expressing HepG2-2-NS4B cells, compared with that of non-induced HepG2-2-NS4B cells. FASTK is a transcriptional regulator that can modulate the alternative splicing of Fas gene and promote the translation of Fas variants with opposite physiological effects. Since the production of soluble Fas isoform may render cells more resistant to Fas-activated apoptosis, it is possible that NS4B protein down-regulates FASTK mRNA expression and affects Fas/FasL signaling pathway. The amount of secreted soluble Fas of HepG2-2-NS4B was 2.5-fold that of the parental HepG2-2 cells; the ratio of secreted soluble Fas to the total amount of Fas produced by HepG2-2-NS4B was 1.4-fold that of the HepG2-2 cells. When cells underwent anti-Fas antibody-induced apoptosis, the cell viability of HepG2-2-NS4B with doxycycline- induction was 1.7-fold that of HepG2-2-NS4B without doxycycline-treatment. Besides, the level of caspase 3 activation of HepG2-2-NS4B was 41 % that of HepG2-2 when caspase 3 activation was examined as another indicator of cells underwent anti-Fas antibody-induced apoptosis. Taken together, the results link the elevation of soluble Fas level to the repression of caspase 3 activity and down-regulation of FASTK mRNA. Signaling pathways other than the FASTK-mediated apoptosis may also contribute to the increased cell viability regulated by the viral NS4B protein. | en |
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dc.description.tableofcontents | 口試委員會審定書............................................................................................................i
中文摘要...........................................................................................................................ii 英文摘要...........................................................................................................................iii 目錄...................................................................................................................................v 圖表目錄.........................................................................................................................viii 第一章 緒論...................................................................................................................1 一、C型肝炎病毒......................................................................................................1 1.1 C型肝炎病毒顆粒及基因體之構造..............................................................1 1.2 C型肝炎病毒之複製......................................................................................1 1.3 C型肝炎相關之肝病變..................................................................................2 二、肝癌.....................................................................................................................3 2.1肝癌致癌因子................................................................................................3 2.2 肝癌發展之可能機制及其與細胞凋亡的關聯..........................................4 2.3細胞凋亡機制................................................................................................5 三、細胞蛋白質Fas受器.........................................................................................6 3.1 Fas、FasL及其訊息傳遞.............................................................................6 3.2 Fas異構物、基因及alternative splicing.......................................................8 四、細胞蛋白質FASTK...........................................................................................9 五、C型肝炎病毒非構性蛋白NS4B....................................................................10 5.1 NS4B的特性及病毒複製...........................................................................10 5.2 NS4B對細胞生理之影響...........................................................................12 5.3 NS4B與細胞蛋白之交互作用...................................................................13 第二章 研究方向.........................................................................................................14 第三章 材料與方法.....................................................................................................15 一、藥品..................................................................................................................15 二、酵素..................................................................................................................16 三、抗體..................................................................................................................16 四、細胞培養液及轉染試劑..................................................................................16 五、套組試劑..........................................................................................................17 六、其它..................................................................................................................17 七、實驗室提供之質體..........................................................................................17 7.1 pcDNA-NS4B-V5HisTopo.........................................................................17 7.2 pHyg-TRE-NS4B-V5.................................................................................17 7.3 pTet-On.......................................................................................................18 7.4 pcDNA-FASTK-V5HisTopo......................................................................18 八、細胞株及細胞培養...........................................................................................18 8.1 HepG2-2細胞株.........................................................................................18 8.2 HepG2-2-NS4B細胞株..............................................................................18 九、DNA轉染.........................................................................................................19 十、細胞全蛋白質之收取.......................................................................................19 十一、蛋白質定量...................................................................................................20 十二、正十二烷硫酸鈉-聚丙醯胺板膠電泳.........................................................20 十三、西方墨點法...................................................................................................21 十四、萃取RNA.....................................................................................................21 十五、反轉錄-聚合酵素鏈反應.............................................................................22 十六、即時定量聚合酵素鏈鎖反應......................................................................22 十七、Fas抗體刺激細胞凋亡................................................................................22 十八、Trypan Blue Exclusion Assay.......................................................................23 十九、Caspase 3活性測定.....................................................................................23 二十、ELISA for sFas.............................................................................................24 第四章 結果.................................................................................................................25 一、HepG2-2-NS4B-V5穩定細胞株的建立及表現............................................25 二、NS4B蛋白質對FASTK基因表現量的影響.................................................25 三、NS4B對Fas活化所導致之細胞凋亡的影響................................................26 3.1細胞活性....................................................................................................26 3.2 Caspase 3 活性.........................................................................................27 四、NS4B蛋白質對Fas異構物表現的影響.......................................................28 第五章 討論.................................................................................................................30 一、穩定細胞株表現NS4B之情形......................................................................30 二、NS4B蛋白質對於FASTK及細胞的可能影響..............................................31 三、NS4B蛋白質對細胞生理之影響...................................................................32 第六章 圖表.................................................................................................................37 第七章 附圖.................................................................................................................44 第八章 參考文獻.........................................................................................................46 | |
dc.language.iso | zh-TW | |
dc.title | C型肝炎病毒非結構性蛋白質NS4B經由細胞FASTK對Fas/FasL訊息傳遞產生的影響 | zh_TW |
dc.title | The Effects of Hepatitis C Virus Nonstructural Protein 4B on Fas/FasL Signaling – Through Fas-Activated Serine/Threonine Kinase (FASTK) | en |
dc.type | Thesis | |
dc.date.schoolyear | 96-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 董馨蓮,林素芳 | |
dc.subject.keyword | NS4B,FASTK,Fas, | zh_TW |
dc.relation.page | 52 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2008-07-31 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 微生物學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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