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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 張鑫 | |
dc.contributor.author | Shing-Tzu Hsu | en |
dc.contributor.author | 許倖慈 | zh_TW |
dc.date.accessioned | 2021-06-15T01:31:38Z | - |
dc.date.available | 2014-10-07 | |
dc.date.copyright | 2011-10-07 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-08-16 | |
dc.identifier.citation | Attwell, S., Roskelley, C., and Dedhar, S. (2000). The integrin-linked kinase (ILK) suppresses anoikis. Oncogene 19, 3811-3815.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42986 | - |
dc.description.abstract | 全球約有1.7億人口感染C型肝炎病毒,其中五分之一的人口可能發展成肝硬化,甚至是肝癌,而全世界肝癌的死亡率更高達第三名。C型肝炎病毒為正股單向RNA病毒,可轉譯出一多蛋白質前驅物,透過宿主及病毒自身的蛋白酶切割成具有不同功能的結構性及非結構性蛋白質。非結構性蛋白質NS3,具有NTPase、RNA helicase及serine protease的功能。先前研究指出,NS3可加速細胞生長及存活於低血清的環境,並可誘發裸鼠產生腫瘤。本實驗室亦證實,NS3在NS4A輔因子存在下,可進行病毒多蛋白質切割及內部截切,並與細胞轉型能力相關。本論文進一步研究發現,當人類肝癌細胞表現NS3蛋白質時,會抑制細胞的貼附能力。已知integrin receptor參與調控細胞的貼附、生長、移動及凋亡,其構型的改變或是表現量的差異,與疾病和腫瘤的發生皆具有極大的關聯性。為了探究NS3是否透過影響integrin參與下游的訊息傳遞,並且影響細胞的貼附能力,實驗首先利用不同細胞外基質觀察細胞貼附能力的差異性,同時收取細胞萃取液進行西方墨點法分析,希望藉此推測NS3蛋白質可能影響的integrin種類。結果發現,不論是以collagen或laminin或fibronectin作為細胞外基質,NS3均會抑制細胞的貼附能力,而且也會造成integrin beta 1總表現量下降的現象。利用共同免疫沉澱及GST pull-down證實NS3蛋白質與integrin beta 1具有交互作用,免疫螢光染色及共軛焦顯微鏡觀察,也發現兩者共存於細胞核周圍的區域。表現NS3蛋白質的次區域時,偵測到NTPase domain及RNA binding domain也會抑制細胞的貼附能力,同時造成integrin beta 1總表現量的下降,但對於活化態的integrin beta 1並無顯著的影響。利用西方墨點法與特異性抗體分析,也發現貼附能力被NS3蛋白質抑制的細胞有ERK表現量上升及FAK表現量下降的趨勢。由此推測,NS3蛋白質可能藉由降低integrin beta 1的表現量,抑制細胞的貼附能力;並可能利用改變integrin beta 1或是下游訊息傳遞因子的表現量及活化程度和位置,進而影響細胞的分化和轉型的特性,而這些現象可能與NS3蛋白質和integrin beta 1具有交互作用相關。這些實驗結果提供了NS3促使細胞癌化的一個可能作用機制。 | zh_TW |
dc.description.abstract | There are 170 million peoples infected with hepatitis C virus in the world. One-fifth of them may develop liver cirrhosis or even liver cancer. The mortality rate of liver cancer is as high as the third. Hepatitis C virus is a positive single strand RNA virus. The viral genome can translate into a polyprotein precursor that is further processed into structural and non-structural proteins by host and viral proteases. The non-structural protein 3(NS3)possesses NTPase, RNA helicase and serine protease activities. Previous studies have demonstrated abilities of the HCV NS3 to enhance growth rate and to allow culture cells to grow in low serum medium. NS3 can also induce tumor formation in nude mice. Our laboratory has earlier demonstrated the roles of NS3 in NS4A-dependent polyprotein processing and internal NS3 cleavage. The internal cleavage is associated with the cell transforming activity of NS3. In the current study, NS3 was shown to inhibit adhesion of Huh7 cells to extracellular matrix. Integrin receptors have previously been demonstrated to be involved in the regulation of cell adhesion, growth, movement, and apoptosis. Changes on their configurations and expression levels correlate with disease progression and cancer formation. To investigate whether NS3 affects integrin downstream signaling pathway and cell adhesion, different extracellular matrixes were first applied to examine the effects of NS3 on cell adhesion. The results demonstrated that NS3 reduced cell adhesion, no matter collagen or laminin or fibronectin was used to the extracellular matrix. In addition, NS3 reduced expression level of total integrin beta 1. Co-immunoprecipitation and GST pull-down assay demonstrated interactions of the NS3 protein with integrin beta 1. Immunofluorescence and confocal microscopy also detected a partial co-localization of NS3 and integrin beta 1 at the perinucleus. The inhibitory effects of NS3 on cell adhesion and integrin beta 1 expression were also observed with the NTPase and the RNA binding domain of the NS3, but no significant change was detected for the active form of integrin. Furthermore, the expression level of FAK was down regulated but ERK was up-regulated in the NS3 expressing cells. Taken together, NS3 may down-regulate the integrin expression causing an inhibitory effect on cell adhesion. Meanwhile, through regulating the expression of integrin and the degree and cellular compartment of activation, NS3 may participate in the integrin signaling pathway, involving in cell proliferation and transforming activity. These results provide a possible mechanism of NS3 in its association with carcinogenesis. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T01:31:38Z (GMT). No. of bitstreams: 1 ntu-100-R98445127-1.pdf: 1339602 bytes, checksum: e53fa97ee570ad21519a59ce168707df (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | 摘要 i
Abstract ii 目錄 iv 圖表目錄 vii 緒論 1 一、C型肝炎病毒的發現與分類 1 二、C型肝炎病毒相關的肝病與治療 1 三、C型肝炎病毒的基因體構造及功能 2 四、非結構性蛋白質NS3的特性 4 五、Integrins訊息傳遞 6 研究目的 11 材料與方法 12 一、藥品 12 二、酵素 14 三、抗體 14 四、細胞培養液及轉染試劑 14 五、套組試劑 15 六、染劑 15 七、其它材料 15 八、細胞株 16 九、實驗室提供的質體 16 十、質體的建構 19 十一、DNA 接合 (Ligation) 19 十二、細菌轉型(Transformation) 20 十三、DNA轉染(DNA transfection) 20 十四、細胞全蛋白質收取 21 十五、蛋白質定量 21 十六、正十二烷硫酸鈉–聚丙醯胺板膠電泳(SDS-polyacrylamide gel electrophoresis, SDS-PAGE) 21 十七、西方墨點法 22 十八、共同免疫沉澱法 23 十九、Glutathione S-transferase (GST)融合蛋白質的製備與GST pull-down分析 23 二十、GST結合分析 24 二十一、細胞黏著度測試 24 二十二、免疫螢光染色(Immuno-fluorescence staining) 25 實驗結果 26 一、NS3蛋白質影響細胞貼附能力 26 二、NS3蛋白質對於內生性integrin beta 1的影響 27 三、NS3和integrin beta 1蛋白質在細胞中分布情形 27 四、NS3蛋白質與次區域和integrin beta 1的交互作用 28 五、NS3蛋白質次區域對細胞貼附能力的影響 29 討論 30 一、NS3蛋白質的表現影響細胞的貼附能力 30 二、表現NS3蛋白質對於細胞內生性integrin beta 1的影響 30 三、NS3和integrin beta 1蛋白質在細胞中分布的位置 31 四、NS3蛋白質與integrin beta 1之間的交互作用 31 五、NS3蛋白質次區域對細胞貼附能力的影響 32 六、NS3蛋白質可能參與的訊息傳遞路徑 32 參考文獻 42 圖表目錄 圖一、表現NS3蛋白質的細胞在不同細胞外基質存在時的貼附情形 34 圖二、表現NS3蛋白質的細胞在不同濃度的細胞外基質存在時的貼附能力 35 圖三、NS3蛋白質表現量的多寡影響內生性integrin beta 1表現量及細胞貼附能力 36 圖四、NS3和integrin beta 1蛋白質在Huh7細胞的分布情形 37 圖五、以共同免疫沉澱法分析NS3與integrin beta 1蛋白質的交互作用 38 圖六、共同免疫沉澱法分析NS3蛋白質次區域與integrin beta 1蛋白質的交互作用 39 圖七、以GST-pull down assay,分析NS3次區域的GST融合蛋白質和integrin beta 1的交互作用 40 圖八、NS3蛋白質次區域對細胞貼附能力的影響 41 | |
dc.language.iso | zh-TW | |
dc.title | C型肝炎病毒非結構性蛋白質NS3對細胞貼附及Integrin訊息傳遞的影響 | zh_TW |
dc.title | The Effects of the Hepatitis C Virus NS3 Protein on Cell Adhesion and Integrin Signaling Pathway | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 沈湯龍,董馨蓮 | |
dc.subject.keyword | C型肝炎病毒,非結構性蛋白質NS3,細胞貼附,integrin, | zh_TW |
dc.subject.keyword | HCV,NS3,cell adhesion,integrin, | en |
dc.relation.page | 47 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-08-16 | |
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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