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???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
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dc.contributor.advisor | 季昭華 | |
dc.contributor.author | Kuo-Wei Huang | en |
dc.contributor.author | 黃國維 | zh_TW |
dc.date.accessioned | 2021-06-13T03:43:25Z | - |
dc.date.available | 2006-07-31 | |
dc.date.copyright | 2006-07-31 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-07-25 | |
dc.identifier.citation | Ashkenazi, A. (2002). Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat Rev Cancer 2, 420-430.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32335 | - |
dc.description.abstract | NK92MI為一種可以快速生長,且可以自行分泌IL-2而不需額外補充的人類自然殺手細胞株,其擁有對多種腫瘤細胞具有毒殺的能力,但卻不會影響到正常的人類細胞,而也由於這些特性,使得此細胞被認為是發展免疫治療絕佳的工具之ㄧ。為了加強NK92MI對抗腫瘤的能力以及拓展其毒殺能力的範圍,在本實驗裡我們將一段崁合受器的基因序列,利用反轉錄病毒系統,將其送入NK92MI內表現。此崁合受器由對癌胚抗原具特異性的單鏈抗體、人類CD8 α hinge region及人類CD3ζ chain transmembrane和intracellular domain所構成。結果在體外細胞毒殺測試方面,可觀察到NK92MI與改造後的NK92MI皆無法對沒有表現癌胚抗原的腫瘤細胞進行毒殺作用;然而實驗發現,原本NK92MI無法毒殺的對象,若其有表現癌胚抗原,則即使在低的作用細胞與目標細胞比例下,改造後的NK92MI即可展現對此細胞高效率且特異的毒殺作用。因此在本實驗中,我們利用崁合受器在NK92MI上的表現,成功的改變了其原本的毒殺能力,而使得其對於表現癌胚抗原的細胞可以展現強大且特異的毒殺作用。若此研究可以持續發展推動,未來有極大的潛力與機會可以用於治療有大量癌胚抗原表現的腫瘤上。 | zh_TW |
dc.description.abstract | The continuously growing and IL-2-independent natural killer (NK) cell line NK92MI is highly cytotoxic against malignant cells of various origins without affecting normal human cells. These properties suggest that NK92MI may be a good candidate as an immunotherapeutic agent. To further enhance the antitumoral activity of NK92MI cells and expand the range of tumor entities suitable for NK92MI–based therapies, here by transduction with a retroviral vector we have generated genetically modified NK92MI cells expressing a chimeric antigen receptor (CAR) specific for the tumor-associated carcinoembryonic antigen (CEA), which is overexpressed in many tumors of epithelial origin. The CAR consists of the CEA-specific single chain antibody (scFv), a flexible hinge region derived from CD8, and transmembrane and intracellular regions of the CD3ζ chain. Transduced NK92MI-scFv (CEA) -ζ cells express significant levels of the fusion protein on the cell surface as determined by fluorescence-activated cell-scanning (FACS) analysis. In cytotoxicity assays, there was no difference in cytotoxic activity of NK92MI and NK92MI-scFv (CEA) -ζ cells toward CEA-negative targets. However, even at low effector-to-target ratios, NK92MI-scFv (CEA) -ζ cells specifically and efficiently lysed CEA-expressing tumor cell lines that were resistant to cytolytic activity of parental NK92MI cells. These results demonstrate that efficient retargeting of NK92MI cytotoxicity can be achieved and might allow the generation of potent cell-based therapeutics for the treatment of CEA-expressing malignancies. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T03:43:25Z (GMT). No. of bitstreams: 1 ntu-95-R93629027-1.pdf: 2023577 bytes, checksum: 174901448d8e0b4f52f4f07f690a0c74 (MD5) Previous issue date: 2006 | en |
dc.description.tableofcontents | 中文摘要…………………………………………………………………V英文摘要……………………………………………………………….VI縮寫符號……………………………………………………….……VII壹、概論……………………………………………………………...1第一章、癌症的免疫療法…………………………………………...1第二章、自然殺手細胞的簡介………………………………...3 第一節、自然殺手細胞的簡介…………..………………………...3
第二節、自然殺手細胞的活化機制………………………………….3 第三節、自然殺手細胞與腫瘤細胞間的關係……………………….5 第三章、自然殺手細胞在腫瘤免疫療法的應用…………………….11第四章、癌胚抗原的簡介…………………………………………….14第五章、研究目標………………………………………….………16 第一節、構築anti-CEA CAR表現載體…….………………………16 第二節、反轉錄病毒系統感染人類NK細胞株(NK92MI)……….17 第三節、活體外測試NK92MI-scFv(CEA)-ζ細胞對表現CEA之人類腫瘤細胞特異的毒殺性….……………………..………17 貳、 材料與方法…………….……..………………………………18第一章、Anti-CEA CAR中各基因序列之建構與合成…………….18 第一節、細胞培養………………..……..………………………...18 第二節、冷凍與解凍細胞…………………..……………………….20 第三節、引子設計……………………………………..…………….21 第四節、西方墨點法…………………………………..…………….22 第五節、RNA的抽取……………………………………...………….25 第六節、反轉錄………………………………………..…………….25 第七節、聚合酵素連鎖反應………………………….……………. 26 第八節、洋菜膠電泳………………………………….……………. 28 第九節、從電泳洋菜膠體中純化DNA…………………………….29 第二章、Anti-CEA CAR的表現載體之構築………….………………31 第一節、T-A cloning…….…………..………………………...31 第二節、大腸桿菌的轉型作用………………..…………………….34 第三節、菌落聚合酵素連鎖反應………..………………………….35 第四節、載體DNA之小量分離法…………………………………...36 第五節、限制脢脢切作用…………………………………..……….37 第六節、載體DNA之定序與基因比對………..…………………….38 第七節、選殖載體的構築…………………………….……………. 38 第八節、反轉錄病毒載體的構築……………………………….…. 42 第九節、大量載體DNA之分離………………………………….…. 46 第三章、Anti-CEA CAR表現載體於哺乳類細胞株之轉染及鍵結活性測試……………48 第一節、種入細胞…………………..…..………………………...48 第二節、表現載體的轉染……………………..…………………….48 第三節、偵測anti-CEA CAR在細胞中的表現…………………….49 第四節、鍵結活性測試………………….……………….………….50 第四章、重組反轉錄病毒的構築及標的細胞的感染…….…………52 第一節、反轉錄病毒包裝細胞的轉染……………………………...52 第二節、標的細胞的感染與篩選…………………..……………….52 第五章、活體外細胞毒殺試驗…………………………….…………54 第一節、偵測anti-CEA CAR在細胞上的表現….…………………54 第二節、活體外細胞毒殺試驗……………………..……………….54 参、 結果…………………………………………………...………57 第一章、Anti-CEA CAR表現載體之構築……………...…………. 57 第一節、製備anti-CEA CAR中各基因序列….……………………57 第二節、建構anti-CEA CAR的表現載體………………………….74 第二章、Anti-CEA CAR表現載體於哺乳類細胞株(BALB/3T3)之轉染及鍵結活性測試……………………………………………… 85 第一節、Anti-CEA CAR在BALB/3T3細胞上的表現………………85 第二節、Anti-CEA CAR之鍵結活性測試………….……………….86 第三章、Anti-CEA CAR於標的細胞(NK92MI)的表現及體外細胞毒殺試驗………………………………………………….……...….87 第一節、NK92MI-scFv(CEA)-ζ細胞的篩……………………..87 第二節、Anti-CEA CAR在NK92MI-scFv(CEA)-ζ細胞的表現…87 第三節、體外NK92MI-scFv(CEA)-ζ細胞之毒殺試驗…………88 肆、討論……………………………………………………………….91 伍、參考文獻……………………………………………………….98 | |
dc.language.iso | zh-TW | |
dc.title | 改造人類自然殺手細胞株使其對表現癌胚抗原之腫瘤細胞具特異之毒殺作用 | zh_TW |
dc.title | Retargeting of human natural killer cell line cytolytic activity to CEA-expressing cancer cells results in specific tumor cell destruction | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 廖光文,季匡華,朱瑞民 | |
dc.subject.keyword | 崁合受器,癌胚抗原,自然殺手細胞, | zh_TW |
dc.subject.keyword | chimeric receptor,carcinoembryonic antigen,NK cell, | en |
dc.relation.page | 104 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2006-07-26 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 獸醫學研究所 | zh_TW |
Appears in Collections: | 獸醫學系 |
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ntu-95-1.pdf Restricted Access | 1.98 MB | Adobe PDF |
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