臺灣博碩士論文加值系統

已知感染C型肝炎病毒和肝細胞癌形成有關。而病毒感染造成的DNA損傷增加以及DNA修復下降，是導致癌症的可能原因之ㄧ。先前研究指出C型肝炎病毒非結構性蛋白質NS3會使細胞產生NO以及ROS並且造成細胞DNA的損傷以及DNA修復能力下降，導致表現NS3的細胞經遺傳毒物 (genotoxic agents)作用後的存活率下降。而NS4A是NS3蛋白酶的輔因子，NS4A也可以增加細胞轉型能力。本實驗使用可誘導NS3-4A表現的穩定細胞株，來探討表現NS3-4A的細胞經過DNA雙股斷裂的遺傳毒物的作用後是否會影響細胞的存活率。MTT細胞存活率的結果顯示，誘導NS3-4A表現會使得細胞在博來黴素 (bleomycin)作用後的存活率下降。接下來探討NS3-4A在遺傳毒物的作用下對DNA損傷修復造成的影響，利用西方墨點法觀察細胞內代表雙股斷裂的γ-H2AX表現量，結果顯示NS3-4A表現會增加細胞經博來黴素處理後的γ-H2AX表現量，代表DNA的損傷修復受到影響，並且γ-H2AX表現量增加的現象具有博來黴素劑量反應關係 (dose response)，另外，結果顯示不管有無誘導NS3-4A，在移除博來黴素後的1小時DNA即修復完畢。最後使用NS3以及缺乏NS3蛋白酶活性的NS3pd-4A穩定細胞株進一步探討，結果顯示NS3以及NS3pd-4A皆會增加博來黴素作用後的γ-H2AX的表現量，因此NS3-4A使細胞在博來黴素的作用下γ-H2AX的表現量增加可能不需要NS4A以及NS3蛋白酶活性的參與。NS3-4A使細胞在博來黴素的作用下存活率下降以及γ-H2AX的表現量增加的確切機制還需要更進一步的研究。

Hepatitis C virus (HCV) infection is associated with the development of hepatocellular carcinoma. Increase of DNA damage and/or decrease of DNA repair caused by viral infection are associated with an increased risk of tumor formation. The HCV nonstructural protein 3 (NS3) was shown to be responsible for the increase of DNA damage and inhibition of DNA repair, mediated by NO and ROS. The effect of NS3 on DNA damage and repair resulted in a decrease in the survival rate of cells response to genotoxic agents. NS4A is a cofactor of NS3 protease. In addition, NS4A facilitates the transforming activity of NS3. In this study, NS3-4A stable cell line was used to investigate whether the expression of NS3-4A would affect cell viability in response to genotoxic agents that generate DNA double strand breaks. The data showed that NS3-4A decreased cell viability in response to bleomycin. To investigate the effect of NS3-4A on DNA damage and repair in response to bleomycin, γ-H2AX level that represents DNA double strand breaks was analyzed by western blotting. The results showed that NS3-4A increased the γ-H2AX level in response to bleomycin in a dose dependent manner. To examine whether the effect of NS3-4A on γ-H2AX level is associated with NS4A or NS3 protease activity, NS3 and NS3 protease activity deficiency NS3pd-4A stable cell lines were used. The data showed that both expressing NS3 and NS3pd-4A increased the γ-H2AX level in response to bleomycin. The results indicated that the effect of NS3-4A on γ-H2AX level is independent of NS4A and NS3 protease activity. Further study is required for the detailed mechanism of NS3-4A-mediated decrease of cell viability and increase of γ-H2AX level in response to bleomycin.

中文摘要………………………………………………………………..……………….i
英文摘要………………………………………………………………….…………….ii
目錄…….……………………………………………………………………………. ...iii
圖表目錄………………………………………………………………….…………….v
緒論...…………………...……………………………………………………..…...…...1
一、C型肝炎病毒 ...………………….....………………………...………….......1
二、C型肝炎病毒的基因體構造及功能…..……………………..…………........1
三、非結構性蛋白質NS3的特性……………….……………………………........4
四、非結構性蛋白質NS4A的特性……………………………..……...……........7
五、C型肝炎病毒與肝細胞癌………..…………………………..………............8
研究目的………………………………………….………………………………........11
實驗材料………………………………………….……………………………………12
一、藥品...………………………….……………………………………...……..12
二、抗體...…………………………………………….…………………...……..14
三、細胞培養液及轉染試劑 ...………………………………………..………..14
四、套組試劑...……………………………….………………….…..…………..14
五、其他材料...……………………………….………………..….………….… 15
六、細胞株...………………………………….………………...…………….… 15
實驗方法………………………………………….……………………………………17
一、 細胞全蛋白質收取...………….…………………………..….……………..17
二、 蛋白質定量…………………………………….………..………………..…17
三、 正十二烷硫酸鈉-聚丙醯胺板膠電泳(SDS-polyacrylamide gel electrophoresis, SDS-PAGE)……………………..…………..………..….17
四、 西方墨點法 (Western blotting)…………………….………...…………..…18
五、 細胞存活率分析 (MTT assay)…………………………………………..…19
六、 γ-游離輻射 (γ-ray)………………………………...…………………..……20
實驗結果………………………………………….……………………………………21
一、 表現NS3-4A對細胞存活率沒有影響…….……………………...……...21
二、 表現NS3-4A，降低細胞經博來黴素作用後的存活率…………………...21
三、 表現NS3-4A，增加細胞經博來黴素作用後的γ-H2AX表現量…………22
四、 表現NS3-4A，不影響細胞經博來黴素作用後的DNA修復………….23
五、 表現NS3或NS(3pd-4A)，增加細胞經博來黴素作用後的γ-H2AX表現量………………………………………………………………………….23
討論……………………………………………….……………………………………25
一、 不同genotoxic agents刺激，NS3-4A對細胞存活率的影響……………….25
二、 NS3-4A對細胞生長調控的影響…………………………………………...26
三、 NS3-4A對細胞DNA損傷以及修復的影響………………………………26
四、 NS3-4A對細胞凋亡的影響………………………………………………...27
圖表……………………………………………….………………………………….. 29
參考文獻…………………………………………………………………….…… 42

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