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研究生:陳彥劦
研究生(外文):Yen-Hsieh Chen
論文名稱:日本腦炎病毒非結構性蛋白質NS3造成細胞死亡及轉型之機制探討
論文名稱(外文):Evaluation of Japanese encephalitis virus NS3 protein to induce apoptosis (HeLa and Vero) and transformation (NIH 3T3).
指導教授:魏秋偉魏秋偉引用關係
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:2007
畢業學年度:95
語文別:中文
論文頁數:65
中文關鍵詞:日本腦炎病毒非結構性蛋白質細胞死亡轉型
外文關鍵詞:Japanese encephalitis virusNS3apoptosistransformation
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日本腦炎病毒(Japanese encephalitis virus; JEV)屬於黃質病毒科黃質病毒屬,所引起的腦炎每年在中國大陸,印度及東南亞等地都有廣泛流行。病毒以接受器媒介的內吞作用進入細胞後進行RNA的複製及聚蛋白的合成,而此聚蛋白中的非結構性蛋白NS3具有絲胺酸蛋白酶(serine protease)、三磷酸水解酶(triphosphatase)、RNA水解螺旋酶(helicase)的活性。針對同科其他病毒的NS3蛋白,在細胞凋亡或細胞轉型都有相關研究。本實驗將全段JEV NS3以及RNA水解螺旋酶區域分別接至自發螢光蛋白表現載體pEGFP-C3,轉染細胞後表現NS3螢光融合蛋白。結果顯示,轉染並表現NS3螢光融合蛋白的HeLa及Vero細胞體積縮小,染色體濃縮。進一步更確認上述細胞表現Caspase-3及Caspase-9的活性,認為NS3以活化Caspase-9的方式誘導上述細胞產生凋亡現象。另外,文獻亦指出,隸屬同科的C型肝炎病毒NS3蛋白具有誘導NIH 3T3細胞轉型之能力,因而將JEV NS3質體轉染NIH 3T3細胞後進行軟凝膠分析(soft-agar assay),結果顯示
轉染細胞產生細胞聚落(cell colony),並確認此類細胞已改變細胞形態。另一方面以G418篩選亦顯示,持續表現NS3蛋白之細胞產生堆疊,不受貼附限制(contact-inhibition),顯示亦成功轉型。因此認為JEV NS3全長蛋白及其RNA水解螺旋酶活性區域對於不同細胞對象具有促使細胞凋亡或轉型的能力。
Previously reports indicated that flaviviral non-structural protein 3
(NS3) displays serine protease, triphosphatase and RNA helicase activities and serveral flaviviral NS3 had further found their ability to triggercaspase-dependent apoptosis. To clarify if Japanese encephalitis virus (JEV) NS3 and its helicase domain alone can induce apoptosis, we applied mophology observation, dye exclusion and caspase activity methods. After liposome-based transfection, JEV NS3-expressing cells exhibit the mophology of apoptotic cells, i.e. cell shrinkage, chromatin condensation, and nuclear fragmentation. Moreover, these cells also show their in situ
caspase-9 and downstream caspase-3 activities. Additionally, former
research had reported that NS3 protein of Hepatitis C virus (HCV) could induce transformation of NIH 3T3 cells. Also, those JEV NS3-expressing 3T3 cells loss contact-inhibition, become ancharage-independent, and shape into cell colonies in soft-agar, which all correspond with the mophology of transformed cells. In conclusion, JEV NS3 protein and its helicase domain are capable of triggering caspase-9-based apoptosis in HeLa and Vero cells and transformation in NIH 3T3 cells.
中文摘要i
英文摘要ii
第一章、緒論1
第一節、日本腦炎與日本腦炎病毒1
壹、日本腦炎及病毒的發現與流行1
貳、日本腦炎病毒的傳播2
參、日本腦炎病毒與病毒複製3
肆、日本腦炎病毒NS3蛋白質之功能5
第二節、細胞凋亡與病毒5
壹、細胞凋亡的特性5
貳、病毒與細胞凋亡8
第二章、材料與方法9
第一節、實驗用細胞株9
第二節、NS3表現載體的製備9
壹、引子的設計與聚合酶連鎖反應9
貳、TA
cloning 10
參、細菌轉型10
肆、轉接至pEGFP-C3載體10
伍、大量製備NS3表現載體11
第三節、NS3表現載體的轉染12
第四節、細胞核染色12
第五節、Caspase-3活性確認12
第六節、Caspase-8活性確認13
第七節、Caspase-9活性確認13
第八節、軟凝膠分析14
第三章、結果15
第一節、GFP-NS3表現質體製備與表現15
第二節、NS3與細胞凋亡之探討15
壹、GFP-NS3螢光融合蛋白的表現與細胞形態觀察15
貳、轉染後細胞核形態觀察16
參、轉染後細胞死亡率之測定16
肆、Caspase活性之測定17
(一)、Caspase-3活性測定17
(二)、Caspase-8活性測定18
(三)、Caspase-9活性測定18
第三節、NS3與細胞轉型之探討19
壹、軟凝膠分析觀察19
貳、Cell
colony
isolation 19
參、G418篩選轉染後之NIH
3T3
cell 20
第四章、討論21
第一節、NS3全長蛋白及水解螺旋酶區域造成細胞凋亡之差
異21
第二節、NS3誘導之細胞凋亡於不同細胞間之差異22
第三節、日本腦炎病毒與其它黃質病毒NS3造成細胞凋亡之
差異23
第四節、NS3全長蛋白及水解螺旋酶區域造成細胞轉型之差
異25
第五節、日本腦炎病毒與C型肝炎病毒NS3造成細胞轉型之差
異26
參考文獻28
圖表33
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