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dc.contributor.advisor | 王萬波 | |
dc.contributor.author | Mei-Ling Kuan | en |
dc.contributor.author | 官玫玲 | zh_TW |
dc.date.accessioned | 2021-06-15T05:59:15Z | - |
dc.date.available | 2011-09-09 | |
dc.date.copyright | 2010-09-09 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-17 | |
dc.identifier.citation | Adams, J. M. & Cory, S. 2001. 'Life-or-death decisions by the Bcl-2 protein family.' Trends Biochem Sci, 26:1, 61-6.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47425 | - |
dc.description.abstract | HER2/neu proto-oncogene is a member of epidermal growth factor receptor family. Overexpression and amplification of HER2/neu is found in many types of human cancer, including ovary, breast, lung, bladder, colon and gastrointestinal cancers. Overexpression of HER2/neu in cancers is correlated with enhanced metastasis, angiogenesis, and chemoresistance, and poor prognosis. Therefore treatment of HER2/neu-overexpressing cancers has become an urgent issue in cancer therapy.
Previously we reported that simian virus 40 (SV40) T/t-common polypeptide, which contains the N-terminal common domain of SV40 large T and small t antigens, could specifically induce apoptosis in HER2/neu-overexpressing human cancer cell lines but not in non-transformed cell lines and HER2/neu low-expressing human cancer cell lines. In this study, we investigated the anti-tumor effect of combination therapy using T/t-common and granulocyte macrophage colony-stimulating factor (GM-CSF), which can enhance the tumor-antigen presentation capacity of dendritic cells and is one of the most potent cytokines used in cancer immunotherapy. Since T/t-common is known to be able to induce apoptosis of HER2/neu-overexpressing cancer cells, it is possible that GM-CSF may enhance the anti-tumor effect of T/t-common by inducing anti-tumor immunity. To test the anti-tumor effect of T/t-common plus GM-CSF in immuno-competent mice bearing the neu-overexpressing MBT-2 tumors, we first tested whether T/t-common could induce apoptosis in neu-overexpressing MBT-2 bladder cancer cells. Our data indicated that infection of adenovirus carrying the T/t-common gene (rAd-T/t) could specifically induce apoptosis in neu-overexpressing MBT-2 cancer cells but not in neu low-expressing mouse colon CT-26 cancer cells. We next tested whether treatment of established MBT-2 tumors in syngenic C3H mice with both rAd-T/t and adenovirus carrying the GM-CSF gene (rAd-GMCSF) could lead to greater tumor suppression than treatment with adenovirus carrying either gene alone. We did not get a conclusive result for this experiment primarily due to two reasons: (i) heterogeneous tumor size caused by MBT-2 cancer cells; and (ii) suppression of anti-tumor immune response caused by adenovirus infection. To solve the first problem (i.e., heterogeneous tumor size), we have cloned the MBT-2 cells and tested the growth of these MBT-2-derived clones in syngenic C3H mice. We obtained three MBT-2-derived neu-overexpressing clones which gave homogeneous tumor size and grew at a rate similar or faster than parental MBT-2 cells. These clones can be used in the future experiments. To solve the second problem (i.e., suppression of immune response by delivering vector adenovirus), We tested the possibility of using positive-charged liposome to deliver genes into MBT-2 tumors grown in immuno-competent C3H mice. Our preliminary data indicated that positive-charged liposome could introduce the green fluorescence protein gene into established MBT-2 tumors through intratumor injection. Further studies are needed to demonstrate whether positive-charged liposome could introduce T/t-common and GM-CSF genes into established MBT-2 tumors in C3H mice and whether liposome carrying these two genes could be used to treat neu-overexpressing MBT-2 tumors in immuno-competent C3H mice. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T05:59:15Z (GMT). No. of bitstreams: 1 ntu-99-R97445109-1.pdf: 2214874 bytes, checksum: f4957bd1b39cab2bcd17e3f3a5c842a8 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 中文摘要……………………………………………………………………...……......i
Abstract………………………………………………………………..…...................iii 目錄…………………………………………………………………....…....................v 圖目錄…………………………………………………………………......................vii 緒論………………………………………………………………….…………...........1 研究目的…………………………………………………………….…….................11 • 實驗材料…………………………………………………….………….........12 一、 化學藥品…………………………………………………………….…….........12 二、 酵素 ……...…………………………………………………………….….........14 三、 套組試劑………………………………………………………………………..14 四、 抗體……………………………………………………………………………..14 五、 其它……………………………………………………………………………..14 六、 重組腺病毒載體………………………………………………………………..15 七、 細胞株…………………………………………………………………………..15 • 實驗方法……………………………………………………………………..16 一、 儲存細胞……………….…………………………………………….………16 二、 勝任細胞的製備 ………………………………………………….…………16 三、 細菌轉形……………………………………………………….…….………17 四、 小量質體製備 (Mini-preparation)………………………………..….………18 五、 大量質體製備 (Large-scale plasmid isolation)………………………...........18 六、 西方墨點分析法…………………………………………………..…………20 七、 細胞免疫螢光色…………………………………………………………..…21 八、 純品系重組腺病毒篩選………………………………………….….………21 九、 高效價病毒製備………….….………………………………………………21 十、 高效價病毒純化…………………………..….………………………...........22 十一、 病毒效價檢測……………………………..……………………...………….23 十二、 酵素連結免疫吸附分析 (ELISA)……………….…….….…………………23 十三、 流式細胞儀分析…………………………………………..…......................24 十四、 動物實驗…………..……..…………………………………........................25 實驗結果………………………………………………………………………........26 一、 rAd-GMCSF感染 MBT-2細胞後可以表現 GM-CSF蛋白質……....……....26 二、 SV40 T/t common與 GM-CSF混合療法在本次實驗條件中對於增強抑 制MBT-2癌細胞腫瘤形成之能力並沒有明顯加成效果...............................26 三、 SV40 T/t common與 GM-CSF混合療法在本次實驗條件中對於增強小 鼠毒殺型 T細胞毒殺 MBT-2癌細胞之能力並沒有明顯效果……………...27 四、 HER2/neu-overexpressing癌細胞 MBT-2之衍生細胞株 (subclone) 其 HER2/neu表現量不一致 …………….……………………………………....28 五、 SV40 T/t common可以抑制HER2/neu-overexpressing癌細胞 MBT-2-sub23之 HER2/neu表現量 ………............................................................................28 六、 SV40 T/t common可專一性地造成 HER2/neu-overexpressing的小鼠膀胱癌 細胞 MBT-2-sub23走向細胞凋亡 ………...………..............……….............29 七、 SV40 T/t common可以影響 MBT-2sub23細胞中 Bcl-2與 Bcl-XL的表現...30 八、 SV40 T/t common可以增加 MBT-2-sub23細胞中 JNK活化的情形……....30 討論…………………………………………………………………...………….....32 圖…………………………………………………………………...……...…..........35 附圖……………………………………………………………………......…..........47 參考文獻…………………………………………………………………..…..........48 圖目錄 圖一、 rAd-GMCSF感染後可以使 MBT-2細胞表現 GM-CSF蛋白質……..…..35 圖二、 rAd-T/t單獨注射以及rAd-T/t+rAd-GMCSF混合注射之小鼠腫瘤生長 情形………………………………………………………………….……....36 圖三、 rAd-GMCSF單獨注射以及rAd-T/t+rAd-GMCSF混合注射之小鼠腫瘤 生長情形 ……………………………………………………………............37 圖四、 rAd-T/t及 rAd-GMCSF混合治療在本次實驗條件中無法提高小鼠 T細胞對 MBT-2細胞之毒殺能力………………………………...…………........39 圖五、 HER2/neu在 MBT-2衍生細胞株第 1-27號、MBT-2母細胞株、NIH-3T3 及 CT-26細胞株的表現量.……………………………………….........…...40 圖六、 MBT-2各衍生細胞株在 syngenic C3H小鼠體內形成腫瘤之生長速率...41 圖七、 rAd-T/t感染可以減少 HER2/neu-overexpressing MBT-2-sub23癌細胞 中 HER2/neu蛋白質的表現量 ………………………………….……........42 圖八、 rAd-T/t感染可以增加 HER2/neu-overexpressing MBT-2-sub23細胞進 入細胞凋亡的比例 …………………………………………….….…..........43 圖九、 rAd-T/t感染不會增加 HER2/neu low-expressing CT-26細胞進入細胞 凋亡的比例…………………………………………………….……...........44 圖十、 rAd-T/t 感染 MBT-2-sub23細胞造成細胞凋亡的染色體聚集現象以及 細胞形態之變化………………………………………….…………..….....45 圖十一、 rAd-T/t common可以影響 MBT-2-sub23細胞中 Bcl-2及 Bcl-XL的 表現…………………………………………………...………………......46 | |
dc.language.iso | zh-TW | |
dc.title | 利用猴病毒四十型 T/t-common合併 GM-CSF治療Neu大量表現的小鼠腫瘤 | zh_TW |
dc.title | Combination Therapy Using SV40 T/t-common and GM-CSF in Neu Overexpressing Tumors | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 賈景山,張鑫 | |
dc.subject.keyword | HER2/neu,SV 40 T/t-common,重組腺病毒,GM-CSF,基因治療, | zh_TW |
dc.subject.keyword | HER2/neu,SV 40 T/t-common,recombinant adenovirus,GM-CSF,gene therapy, | en |
dc.relation.page | 52 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-08-17 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 微生物學研究所 | zh_TW |
Appears in Collections: | 微生物學科所 |
Files in This Item:
File | Size | Format | |
---|---|---|---|
ntu-99-1.pdf Restricted Access | 2.16 MB | Adobe PDF |
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