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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張鑫(Shin C. Chang) | |
| dc.contributor.author | Chung-Hsin Shih | en |
| dc.contributor.author | 史宗鑫 | zh_TW |
| dc.date.accessioned | 2021-06-15T02:37:48Z | - |
| dc.date.available | 2014-09-15 | |
| dc.date.copyright | 2009-09-15 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-08-12 | |
| dc.identifier.citation | Alter, H. J., R. H. Purcell, P. V. Holland, and H. Popper. 1978. Transmissible agent in non-A, non-B hepatitis. Lancet 1 (8062):459-463.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44050 | - |
| dc.description.abstract | C型肝炎病毒 (Hepatitis C virus)為正向單股RNA病毒，在進入細胞之後可利用其正股RNA基因體直接轉譯成多蛋白質 (polyprotein)，再經由細胞內部的胜肽酶 (peptidase)以及病毒自身的蛋白酶 (protease)切割成具有不同功能的結構性蛋白質以及非結構性蛋白質。非結構性蛋白質NS3，全長631個胺基酸，具有蛋白酶和RNA解旋酶 (RNA helicase)的活性；在病毒複製的過程中，主要扮演將多蛋白質切割成各個蛋白質單元以及幫助解開雙股RNA的功能。在先前的研究中，發現C型肝炎病毒基因型1b 的NS3蛋白質具有內部截切 (internal cleavage)的活性，又以IPT402︱S為主要的截切位點。相較於NS3全長蛋白質，內部截切後的N端產物NS3(1-402)蛋白質，具有較強的細胞轉型能力 (transforming activity)。先前的研究亦發現NS3蛋白質具有加快細胞生長、使細胞能在低血清環境下生長的能力。在穩定表現的HepG2細胞中，NS3可活化JNK以及MAPK訊息傳遞。為了要進一步瞭解NS3在C型肝炎病毒致病機轉上的角色，本研究一方面利用tandem affinity purification的系統分析可以和NS3(1-402)共同純化的細胞因子。另一方面，利用短暫轉染和酵母菌雙雜合系統，探討NS3對細胞凋亡與focal adhesion訊息傳遞的影響。結果發現NS3(1-402)可以降低anoikis形式的細胞凋亡，並可以造成Huh-7細胞內部FAK蛋白質量的上升。另外NS3(1-402)會提高293細胞中focal adhesion訊息傳遞路徑上c-src(Y418)以及paxillin(Y118)磷酸化的程度。推測NS3蛋白質可能藉由N端的區域活化focal adhesion訊息傳遞來造成細胞轉型。 | zh_TW |
| dc.description.abstract | Hepatitis C virus is a positive-sense single-stranded RNA virus. After entry into cells, the virus synthesizes its polyprotein precursor from the viral genomic RNA. The polyprotein precursor can be further processed by cellular peptidase and viral proteases into different structural and nonstructural proteins. The nonstructural protein 3 (NS3) consists of 631 amino acids and possesses protease and RNA helicase activities. The NS3 protein plays major roles in the polyprotein processing and RNA unwinding during viral multiplication. In pervious studies, NS3 protein of genotype 1b was found to have internal cleavage activity, and IPT402|S is the major cleavage site. The N-terminal cleavage product, NS3(1-402) has a higher transforming activity than that of the full length NS3 protein. Studies also demonstrated an enhanced growth rate and ability to grow at low concentration of serum in the presence of NS3. In addition, JNK and MAPK signaling pathways were activated in HepG2 cells stably expressing NS3. Recent studies also suggest an association of focal adhesion with tumor formation. Towards understanding the roles of NS3 in the pathogenesis of HCV, tandem affinity purification system was used to analyze cellular factors that interact with NS3(1-402). On the other hand, transient transfection and yeast-two hybrid experiments were performed to examine the effects of NS3 on cell apoptosis and focal adhesion signaling. The results demonstrated a decrease of anoikis apoptosis and a slightly increased level of FAK in Huh-7 cells expressing NS3(1-402). In addition, NS3(1-402) protein enhanced the phosphorylation of both c-src(Y418) and paxillin(Y118) in 293 cells. Taken together, these results suggest that the N-terminal domain of HCV NS3 protein may interfere focal adhesion signaling. Activation of focal adhesion signaling pathway may correlate with the transforming activity of NS3 protein. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T02:37:48Z (GMT). No. of bitstreams: 1 ntu-98-R96445123-1.pdf: 2007048 bytes, checksum: e1e419ef3dfcba5cd6e18ac3950d65a4 (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 研究方向.............................................................................................................13
材料與方法.........................................................................................................14 一、藥品........................................................................................................14 二、酵素........................................................................................................15 三、抗體........................................................................................................15 四、細胞培養液及轉染試劑............................................................................16 五、套裝試劑.................................................................................................16 六、其他.........................................................................................................17 七、細胞株.....................................................................................................17 八、實驗室提供的質體...................................................................................17 九、質體的構築.............................................................................................19 十、聚合酶連鎖反應 (Polymerase chain reaction, PCR)………..……..……21 十一、DNA接合 (Ligation).............................................................................21 十二、DNA轉型 (Transformation) ................................................................21 十三、DNA轉染 (DNA transfection)..............................................................22 十四、Tandem affinity purification………………….....…………………………22 十五、蛋白質定量...........................................................................................23 十六、正十二烷硫酸鈉-聚丙醯胺板電泳 (SDS-polyacrylamide gel electrophoresis, SDS-PAGE)..........................................................23 十七、西方墨點法 (Western blot analysis)....................................................24 十八、酵母菌轉型 (Yeast transformation)與酵母菌雙雜合 (Yeast two-hybrid)………………...................................................................25 十九、Anoikis細胞凋亡實驗……………………………..……………………….27 二十、流式細胞儀分析 (Flow cytometry)..….……….………………………28 結果…………………………………………………………….………………………29 一、建立Tandem affinity purification系統以及蛋白質純化…….….......………29 二、Integrin-beta1、parvin、zyxin酵母菌雙雜合實驗………………..………29 三、293細胞的anoikis細胞凋亡…………………………………………….…..30 四、NS3蛋白質及其片段在Huh-7以及293細胞表現情形…………..………..31 五、p53蛋白質在NS3相關片段轉染293細胞表現情形………….……………31 六、FAK蛋白質在293以及Huh-7細胞內部的表現情形………………………32 七、NS3蛋白質對paxillin蛋白質Tyr118位置磷酸化的影響……………….32 八、src蛋白質在表現NS3蛋白質的293細胞內部的表現情形………………..32 討論…………………………………………………………………………….………34 一、酵母菌雙雜合實驗結果……………………………………………….………34 二、NS3蛋白質在不同細胞內表現的情形………………………………...…….34 三、造成FAK蛋白質增加以及focal adhesion訊息傳遞活化的可能機制..….34 四、活化ERK以及JNK訊息傳遞路徑…………………………………….……..36 五、Focal adhesion訊息傳遞在293以及Huh-7細胞的差異…..…….……..36 圖表…………………………………………………………………………………….38 參考文獻…………………………………………...……………...…………………..48 | |
| dc.language.iso | zh-TW | |
| dc.subject | 焦點黏著訊息傳遞 | zh_TW |
| dc.subject | 細胞轉型 | zh_TW |
| dc.subject | NS3蛋白質 | zh_TW |
| dc.subject | C型肝炎病毒 | zh_TW |
| dc.subject | 黏著斑訊息傳遞 | zh_TW |
| dc.subject | focal adhesion | en |
| dc.subject | NS3 | en |
| dc.subject | HCV | en |
| dc.subject | transforming activity | en |
| dc.title | C型肝炎病毒NS3蛋白質對焦點黏著訊息傳遞之調控 | zh_TW |
| dc.title | Regulation of Focal Adhesion Signaling by the NS3 Protein of Hepatitis C Virus | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳美如(M.R. Chen),林淑華(S.W. Lin) | |
| dc.subject.keyword | C型肝炎病毒,NS3蛋白質,細胞轉型,焦點黏著訊息傳遞,黏著斑訊息傳遞, | zh_TW |
| dc.subject.keyword | HCV,NS3,transforming activity,focal adhesion, | en |
| dc.relation.page | 55 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-08-12 | |
| 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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