Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36221
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dc.contributor陳志平zh_TW
dc.contributor姜中人zh_TW
dc.contributor.advisor曾志正zh_TW
dc.contributor.author王信富zh_TW
dc.contributor.authorWang, Hesin-Fuen_US
dc.contributor.other中興大學zh_TW
dc.date2010zh_TW
dc.date.accessioned2014-06-06T07:54:14Z-
dc.date.available2014-06-06T07:54:14Z-
dc.identifierU0005-2307200916420400zh_TW
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(1986) The neu oncogene encodes an epidermal-growth-factor receptor-related protein. Nature 319:226—230. 27. Barnes, M. N., Deshane, J. S., Siegal. G. P., Alvarez, R. D. and Curiel, (1996) Novel gene therapy strategy to accomplish growth factor modulation induces enhanced tumor cell chemosensitivity. Clin. Cancer Res. 2:1089-1095. 28. Baselga J, Tripathy D, Mendelsohn J, Baughman S, Benz CC, Dantis L, Sklarin NT, Seidman AD, Hudis CA, Moore J, Rosen PP, Twad-dell T, Henderson IC, Norton L. (1996) Phase II study of weekly intravenous recombinant humanized anti-p185 HER2 monoclonal an-tibody in patients with HER2/neu-overexpressing meta 29. Baselga, J., Norton, L., Albanell, J., Kim, Y. M. and Mendelsohn, J. (1998) Recombinant humanized anti-HER2 antibody (Herceptin) enhance the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts. Cancer Res. 58:2825-2831. 30. Baselga, J., Seidman, A. D., Rosen, P. P. and Norton, L. (1997) HER2 overex-pression and paclitaxel sensitivity in breast cancer: therapeutic implications. Oncology 11:43—48. 31. Beaudoin, F. and Napier, J. A. (2000) The targeting and accumulation of ectopically expressed oleosin in non-seed tissues of Arabidopsis thaliana. Planta 210: 439-445. 32. Beerli, R. R., Graus-Porta, D., Woods-Cook, K., Chen, X. M., Yarden, Y., Hynes, N. E. (1995) Neu differentiation factor activation of ErbB-3 and ErbB-4 is cell specific and displays a differential requirement for ErbB-2. Mol Cell Biol 15:6496—6505. 33. Benz, C. C., Scott, G. K., Sarup, J. C., Johnson, R. M., Tripathy, D., Coronado, E., Shepard, H. M. and Osborne, C. K. (1992) Estrogen-dependent, tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected with HER2/neu. Breast Cancer Res. Treat. 24:85-95. 34. Chaudhary, S., Parmenter, D. L. and Moloney, M. M. (1998) Transgenic Brassica carinata as a vehicle for the production of recombinant proteins in seeds. Plant Cell Report 17: 195-200. 35. Chen, E. C. F., Tai, S. S. K., Peng, C. C. and Tzen. J. T. C. (1998) Identification of three novel unique proteins in seed oil bodies of sesame. Plant Cell Physiol. 39: 935-941. 36. Chen, J. C. F., Lin, R. H., Huang, H. C. and Tzen, J. T. C. (1997) Clon- ing, expression and isoform classification of a minor oleosin in sesame oil bodies. J. Biochem. 122: 819-824. 37. Chen, J. C., Tsai C. C. Y. and Tzen J. T. C. (1999) Cloning and secondary structure analysis of caleosin, a unique calcium-binding protein in oil bodies of plant seeds. Plant Cell Physiol. 40: 1079-1086. 38. Ching, T. M. (1970) Glyoxysomes in megagametophyte of germination Ponderosa pine seeds. Plant Physiol. 46: 475-482. 39. Donalson, R. P. and Beevers, H. (1977) Lipid composition of organelles from germinating caster bean endosperm. Plant Physiol. 59: 259-263. Frey-Wyssling, A., Grieshaber and E., Muhlethaler, K. (1963) Origen of 37.spherosomes in plant cells. J. Ultrastruc. Res. 8: 506-516. Germmrich, A. R. (1981) Ultrastructural and enzymatic studies on the development of microbodies in germinating spores of the fern Anemia phyllitidis Z. Pflanzenphysiol. 102: 69-80. 40. Gurr, M. I. (1980) The biochemistry of triacylglycerol in The Biochemistry of Plants (Stumpf, P. K. and Conn, E. E., eds.) Vol. 4, pp. 204-248. Aca- demic Press, New Yorkzh_TW
dc.identifier.urihttp://hdl.handle.net/11455/36221-
dc.description.abstract植物種子儲存三酸甘油酯(triacylglycerol) 做為發芽所需之量。三酸甘油酯儲存於一種植物種子特有的胞器稱為油體。目前所提出的油體構造模型是一團三酸甘油酯包在一層磷脂質(phospholipid)內,此磷脂質層鑲滿了構造蛋白質叫油體膜蛋白(oleosin)及一些微量的蛋白質。 在本研究中,我們將oleosin與一個具有特殊功用的胜肽融合,以大腸桿菌大量生產此融合蛋白質,接著運用油體重組技術製作人造油體。經由不同pH值、油種及油種用量等條件檢測,得知以芝麻油建構之人造油體在pH 7.5時之穩定度最佳、且顆粒大小一致。最後,我們將所建構的人造油體與腫瘤細胞株作用,結果顯示此人造油體可以專一的與腫瘤細胞結合。zh_TW
dc.description.abstractPlant seeds store triacylglycerols (TAGs) as energy sources for germination and postgerminative growth of seedlings. The storage TAGs are confined to discrete spherical organelles called oil bodies. An oil body contains a TAG matrix surrounded by a monolayer of phospholipids (PLs) embedded with abundant oleosins and some minor proteins of higher molecular mass. In this study, oleosin was fused with a peptide with a special function. The fused protein was overproduced n in Escherichia coli and then utilized for reconstitution of artificial oil bodies (AOBs). After a systematic investigation, AOBs made of sesame oil at pH 7.5 were found to exhibit the highest stability with a size distribution of homology. Finally, after co-incubation of AOBs with several lines of tumor cells, the result shows that the AOBs elicit the ability to bind to tumor cells in a very specific manner.en_US
dc.description.tableofcontents目 次 中文摘要………………………………………………………………ii 英文摘要………………………………………………………………iii 目 次………………………………………………………………iv 圖 目 錄………………………………………………………………vii 表 目 錄………………………………………………………………ix 第一章 緒論…………………………………………………………11 第二章 文獻回顧……………………………………………..……12 2.1 HER-2/neu 基因的簡介…………………………………………12 2.2 Affbody ZH®的介紹與應用之簡介………………………..……17 2.3 油體 (Oil body)…………………………………………………18 2.4 油體膜蛋白 (Oleosin)…………………………………..……20 第三章 實驗材料與方法……………………………………………24 3.1重組質體之建構pJOl-olel-ZH2………………………...………24 3.2 菌種收集方法………………………………………………...…26 3.3 融合蛋白萃取……………………….…………………..………26 3.4 oleZH2融合蛋白建構油體油體(AOB)最適化條件製備……….…26 3.4.1油和融合蛋白比例建構油體(AOB)………...…...……………… 26 3.4.2不同pH 條件建構油體(AOB)…..……..……...………………… 27 3.4.3不同油種建構油體(AOB)………………………………...............27 3.5人造油體(AOB)檢測………..………………………………………28 3.5.1 渾濁度測試為了人造油體(AOB)穩定度…………………….....28 3.5.2 油體(AOB)粒徑分析………………….……………………........28 3.6細胞測試………………………..……….………………………......29 3.6.1 細胞株的培養………………….……………………...................29 3.6.2油溶解不同濃度螢光染劑(Yellow GGK) 和融合蛋白建構油體(AOB)………..……………...…………………………………………..29 3.6.3 油體上融合蛋白質(oleZH2)對癌細胞之專一性測試…….……29 3.6.4 油體上融合蛋白質(ZH2)對活細胞作測試……………………..30 第四章 實驗結果…...…………...................................................…......32 4.1發展油體載體系統……………….……………...…………………32 4.1.1以大腸桿菌生產融合蛋白”oleZH2 ”.............................................32 4.1.2芝麻油和融合蛋白質量(oleZH2)測試出最佳的比例………...…32 4.1.3不同pH找出油體最合適的pH值………………………..............35 4.1.4運用各種來源油建構油體測試出最合適的油…………….….....38 4.2油體上融合蛋白質(ZH2)對各種癌細胞之專一性測試……...……40 4.2.1油體對固定細胞(MCF7和MCF7/Her18)作測試……………..…40 4.2.2不同MOI的油體對活細胞(MCF7、MCF7/Her18和SKOV3)作測試……………………………………….................................…………41 4.2.3油體與細胞(MCF7、MCF7/Her18和SKOV3) 作用不同時間點的測試……………………….....................................................................44 第五章 討論未來與展望…………………………………………......50 第六章 參考文獻…………………………………………………..…51 第七章 附錄……………………………………………………..……54 7.1 菌種之儲存與馴養………………………………………………...54 7.1.1 菌種儲存……………………………………………..……….….54 7.1.2 菌種馴養………………………………………………………....54 7.1.3 培養機的配製…………………………………………………..54 7.2 DNA 純化方法……………………………………………………..55 7.2.1 質體純化……………………………….………………….……55 7.2.2 瓊脂凝膠萃取DNA 片段…………………………………..….56 7.2.3 PCR DNA 純化……………………….…………………….….57 7.2.4 DNA濃度測量…………………..……………………………...57 7.3 剪切反應、連接反應、凝膠電泳法…………………………….…58 7.3.1 剪切反應………………………………………………………..58 7.3.2 凝膠電泳法………………………………………………….….59 7.3.3 連接反應……………………………………..……………….….60 7.4 化學法製備勝任細胞……………………………………………...60 7.5 轉殖作用………..…………………………………………….……61 7.6 聚合酵素連鎖反應…………………….……………………..........61 7.7 蛋白質電泳..…………………………..……………………...……62 圖 目 錄 1. 圖2.1 表皮生長因子接受器家族(EGF receptor family)…..........…14 2. 圖2.2 p185 結構…………….........................................…….....…15 3. 圖2.3 HER2/neu蛋白質在細胞內的訊號傳導……………….....…16 4. 圖2.4油體的結構…………………………………………..…….…20 5. 圖3.1質體pJO1-ole-ZH2 建構流程結構圖…………………….…25 6. 圖4.1以BL21(DE3)/pJO1-oleZH2重組菌株生產油體融合蛋白的蛋白質電泳膠分析圖……………………………………...……………...32 7. 圖4.2油和融合蛋白比例建構油體過程所觀察到的現象………...33 8. 圖4.3油和融合蛋白比例建構油體的顯微鏡觀察………………...34 9. 圖4.4油和融合蛋白比例建構油體的粒徑分析圖………….……..34 10. 圖4.5油和融合蛋白比例建構油體的穩定度測試圖…………….35 11. 圖4.6 不同pH條件建構油體過程所觀察到的現象…………..…36 12. 圖4.7 不同pH條件建構油體時顯微鏡觀察……………...……...36 13. 圖4.8 不同pH條件建構油體的粒徑分析圖…..............................37 14. 圖4.9 不同pH條件建構油體的穩定度測試圖圖………………..37 15. 圖4.10各種油種類建構油體過程所觀察到的現象……………...38 16. 圖4.11各種油種類建構油體時顯微鏡觀察………………...........39 17. 圖4.12各種油種類建構油體的粒徑分析圖.……………………..39 18. 圖4.13各種油種類建構油體的穩定度測試圖…………….……..40 19. 圖4.14油體對固定細胞MCF7和MCF7/Her18專一測試的螢光顯微鏡照片……………………………………………...………….……..41 20. 圖4.15不同MOI的油體對細胞MCF7專一測試的螢光顯微鏡照片…………………………...…………………………………….……..42 21. 圖4.16 不同MOI的油體對細胞MCF7/Her18專一測試的螢光顯微鏡照片…………………………………………………...…….……..42 22. 圖4.17 不同MOI的油體對細胞MCF7/Her18專一測試的共軛螢光顯微鏡照片………………………………………………….…...…..43 23. 圖4.18 不同MOI的油體對細胞SKOV3專一測試的螢光顯微鏡照片……………………………………………………………....……..43 24. 圖4.19 不同MOI的油體對細胞SKOV3專一測試的共軛螢光顯微鏡照片……………………………………………………...….……..44 25. 圖4.20 油體對細胞MCF7作用不同時間點測試的螢光顯微鏡照片………………………………………………………………………..45 26. 圖4.21 油體對細胞MCF7/Her18作用不同時間點測試的螢光顯微鏡照片………………………………………………...……….……..46 27. 圖4.22 油體對細胞MCF7/Her18作用不同時間點測試的共軛螢光顯微鏡照片………………………………………...………….……..47 28. 圖4.23 油體對細胞SKOV3作用不同時間點測試的螢光顯微鏡照片………………………………………………………...……….……..48 29. 圖4.24 油體對細胞SKOV3作用不同時間點測試的共軛螢光顯微鏡照片……………………………………………………...…….……..49 表 目 錄 1. Table 3.1 油和融合蛋白比例….................................…….........….27 2. Table 3.2 粒徑分析儀條件表….............................………….....….28 3. Table 3.3 10X Phosphate-buffer Saline………..................…......….30 4. Table 3.4 3.7% Formaldehyde…....................................……….......30 5. Table 7.1 Luria-Bertain(LB)培養基之組成成份………………......54 6. Table 7.2 TE Buffer 配製………………………………….....……57 7. Table 7.3 剪切反應時各物質加入量…………………….....……..59 8. Table 7.4 5X TBE Buffer 配方…………………………….....……59 9. Table 7.5 6X DNA Loading Buffer配方……………………......….59 10. Table 7.6 連接反應時各物質加入量……………………….........60 11. Table 7.7 PCR Amplification Conditions-1………………….....…62 12. Table 7.8 PCR Amplification Conditions-2……………………….62 13. Table 7.9 蛋白質電泳膠配方(10%)…………………………...…63 14. Table 7.10 LacZ蛋白質電泳膠配方(8%)…………………..……63 15. Table 7.11 蛋白質電泳緩衝溶液配方……………………............63 16. Table 7.12 蛋白質電泳鑄膠用隔絕液…………………………...63 17. Table 7.13 蛋白質電泳呈色劑…………………………………...64 18. Table 7.14 膠片保存液…………………………………………...64 19. Table 7.15 2X Sample buffer……………………………………...64 20. Table 7.16 沈降劑(Lading buffer)………………………………...64 21. Table 7.17 鑄膠用沖洗液……………………………….………..64 22. Table 7.18 去色劑…………………………………….…………..64zh_TW
dc.language.isoen_USzh_TW
dc.publisher生物科技學研究所zh_TW
dc.relation.urihttp://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2307200916420400en_US
dc.subjectoil bodyen_US
dc.subject油體zh_TW
dc.subjecttumor cellsen_US
dc.subject腫瘤細胞zh_TW
dc.title以自營性螢光油體偵測腫瘤細胞zh_TW
dc.titleDetection of tumor cells by self-fluorescent oil bodiesen_US
dc.typeThesis and Dissertationzh_TW
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item.fulltextno fulltext-
item.cerifentitytypePublications-
item.languageiso639-1en_US-
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypeThesis and Dissertation-
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