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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 董馨蓮(Shin-Lian Doong) | |
| dc.contributor.author | Chia-Ming Yang | en |
| dc.contributor.author | 楊嘉銘 | zh_TW |
| dc.date.accessioned | 2021-06-13T07:56:20Z | - |
| dc.date.available | 2005-08-02 | |
| dc.date.copyright | 2005-08-02 | |
| dc.date.issued | 2005 | |
| dc.date.submitted | 2005-07-22 | |
| dc.identifier.citation | 捌、參考文獻
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/36300 | - |
| dc.description.abstract | 第一型人類嗜T細胞病毒(HTLV-1)被認為是造成成人型T細胞白血病(adult T cell leukemia, ATL)及其相關之脊髓病變/熱帶痙攣性下身輕癱(HTLV-1-assoicated myelopathy/tropical spastic paraparesis, HAM/TSP)的主要病原體。HTLV-1 Tax蛋白質因為可以廣泛影響細胞功能被認為是HTLV-1造成疾病的重要致病因子。Tax透過CREB/ATF、SRF和NF-κB相關途徑調控病毒和細胞基因表現。
本實驗試圖評估以GFPTax及其突變蛋白質研究Tax對細胞生長影響的可行性。為了方便在螢光顯微鏡下觀察及便於偵測我們將Tax融合green fluorescence protein (GFP)。本實驗所使用的三種Tax突變蛋白質分別是TaxH43Q(無法活化NF-κB)、TaxS274A(無法活化NF-κB及CREB/ATF)和TaxL320G(無法活化CREB/ATF)。 首先我們調查融合GFP是否會改變Tax轉活化能力及在細胞內位置。將GFPTax及GFPTax突變基因(GFPTaxH43Q, GFPTaxS274A及GFPTaxL320G )構築至受doxycyclin誘導表現質體。利用北方墨點法和西方墨點法分析確認融合蛋白質如預期表現。為確定GFPTax及GFPTax突變蛋白質轉活化能力，進行reporter assay，分別使用pNF-κB-Luc(偵測NF-κB活化能力)及pTxRE-Luc(含有五段重複HTLV-1 LTR 之Tax-responsive element，偵測CREB/ATF活化能力)。令人意外發現GFPTax雖然保有活化NF-κB pathway能力但融合GFP後卻失去活化CREB/ATF pathway能力。TaxH43Q不具活化NF-κB pathway能力，然而GFPTaxH43Q活化NF-κB pathway能力卻被回覆了。利用共軛焦顯微鏡觀察融合GFP之後Tax在細胞內分佈是否和原先不同。TaxS274A和GFPTaxS274A大部分皆均勻分佈在細胞質中。GFPTax、GFPTaxH43Q和GFPTaxL320G主要以點狀分佈於細胞核膜附近。Tax、TaxH43Q及TaxL320G位於細胞核內呈點狀分佈。所以融合GFP確實會改變Tax轉活化能力和細胞內之分佈。 同時我們利用lentivirus為載體系統將GFPTax及GFPTax突變基因送入肝胚瘤細胞株-HepG2和白血病T細胞-Jurkat中表現，觀察GFPTax及突變蛋白質對細胞倍增時間和細胞群落生成能力的影響。但轉染效率只有EGFP和GFPTax達到80%以上，GFPTaxH43Q、GFPTaxS274A和GFPTaxL320G轉染效率只有50%。GFPTax在Hep G2和Jurkat細胞會增加細胞倍增時間，表現GFPTax的Hep G2細胞其細胞群落生成能力下降。表現其餘三種GFPTax突變蛋白質的細胞其生長能力沒有明顯化。綜合上述結果，對於GFPTax及其突變蛋白質是否為研究Tax對細胞生長影響的良好工具還有不確定性，相關問題及解決方法將會在內文中討論。 | zh_TW |
| dc.description.abstract | HTLV-1 is the etiological agent of adult T cell leukemia (ATL), the neurological disease (HTLV-1-assoicated myelopathy/tropical spastic paraparesis, HAM/TSP) and other clinical disorders. The HTLV-1 encoded protein, Tax, affects a variety of cellular functions, prompting it to be considered as the pivotal factor in the process leading to these diseases. Tax modulates the expression of many viral and cellular genes through the CREB/ATF-, SRF-, and NF-κB -associated pathways.
In this study, we would like to evaluate the potential of using GFPTax to examine the Tax-mediated cellular growth. Tax was fused with green fluorescence protein (GFP) for better visualization under fluorescence microscope and detection. The three Tax mutants utilized in this study were TaxH43Q (defect in NF-κB activating ability), TaxS274A (defect in both NF-κB and CREB/ATF activating ability), and TaxL320G (defect in CREB/ATF activating ability). In the first part of the study, we investigated if fusion with GFP would change the cellular distribution and trans-activating function of Tax. Fused GFPTax and GFPTax mutant genes ( GFPTaxH43Q , GFPTaxS274A, and GFPTaxL320G) were cloned into doxycyclin -inducible expression vector respectively. Northern blot and Western blot analysis confirmed the expression of these fusion products as expected. Reporter assay using pNF-κB-Luc (for detection of NF-κB activating ability) and pTxRE-Luc (containing 5 tandem repeats of Tax-responsive element from HTLV-1 LTR, for detection of CREB-activating ability) was performed to evaluate the trans-activating ability of these fusion products. Surprisingly, while GFPTax retained the ability to activate the NF-κB pathway as Tax, the fusion of GFP diminished the Tax-mediated CREB- activating ability. TaxH43Q was shown to be defective in the NF-κB pathway, however, GFPTaxH43Q resumed its NF-κB activating ability. Confocal microscope was utilized to examine the cellular distribution of these fusion products as compared to their authentic proteins. Both TaxS274A and GFPTaxS274A were evenly and predominantly distributed in the cytoplasm. GFPTax, GFPTaxH43Q and GFPTaxL320G were found to be localized near the nuclear membrane in punctuated form. Tax, TaxH43Q and TaxL320G were mainly distributed in the nucleus in punctuated form. The fusion with GFP did affect the cellular distribution and trans-activating function of Tax. Meanwhile, lentivirus-base expression system was employed to transduce the fused GFPTax and mutant genes into the hepatoblastoma cell line – Hep G2 and T-cell leukemia cell line – Jurkat to examine their effects on population doubling time and colony formation ability. The transduction efficiency of EGFP and GFPTax could reach 80%, however, transduction efficiency of GFPTax mutants could only up to 50%. GFPTax increased the population doubling time in Hep G2 and Jurkat cells. Colony formation ability decreased in Hep G2 cells with expression of GFPTax. No obvious cellular growth changes were observed in Hep G2 cells with expression of the three GFPTax mutants. Taken together, our results raised an uncertainty in utilizing GFPTax for investigation on Tax-mediated effects. Problems and solutions will be discussed in the text. | en |
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| dc.description.tableofcontents | 目錄
英文摘要……………………………………………………………………Ⅰ中文摘要……………………………………………………………………Ⅲ 目錄…………………………………………………………………………Ⅳ 壹、 緒論…………………………………………………………………1 一、Human T-cell leukemia virus type 1 (1) HTLV-1基因體構造及基因表現………………………………………1 (2) HTLV-1生活史…………………………………………………………2 二、Tax (1) Tax之結構及生物功能…………………………………………………2 (2) Tax對細胞訊息傳遞途徑的影響…………………………………… 3 (3) Tax對細胞轉形的影響……………………………………………… 5 (4) Tax對細胞週期的影響……………………………………………… 6 (5) Tax對細胞凋亡的影響………………………………………………… 7 (6) Tax對DNA修復的影響……………………………………………… 7 貳、實驗目的………………………………………………………………9 參、材料與方法………………………………………………………………10 肆、結果……………………………………………………………………21 伍、討論……………………………………………………………………26 陸、結果圖表………………………………………………………………31 柒、附錄圖表………………………………………………………………42 捌、參考文獻………………………………………………………………52 | |
| dc.language.iso | zh-TW | |
| dc.subject | Tax | zh_TW |
| dc.subject | 細胞生長 | zh_TW |
| dc.subject | Tax | en |
| dc.subject | cell proliferation | en |
| dc.title | 評估利用GFPTax及其突變蛋白質探討Tax引發效應之可行性 | zh_TW |
| dc.title | Evaluation on the Feasibility of Using GFPTax and Mutants for Investigation on Tax-Mediated Effects | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 93-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳美如(Mei-Ru Chen),楊雅倩(Ya-Chien Yang) | |
| dc.subject.keyword | Tax,細胞生長, | zh_TW |
| dc.subject.keyword | Tax,cell proliferation, | en |
| dc.relation.page | 58 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2005-07-25 | |
| 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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| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-94-1.pdf 未授權公開取用 | 834.08 kB | Adobe PDF |
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