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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 余榮熾 | |
| dc.contributor.author | Shu-Chen Hsieh | en |
| dc.contributor.author | 謝淑楨 | zh_TW |
| dc.date.accessioned | 2021-06-13T07:59:09Z | - |
| dc.date.available | 2006-07-27 | |
| dc.date.copyright | 2005-07-27 | |
| dc.date.issued | 2005 | |
| dc.date.submitted | 2005-07-22 | |
| dc.identifier.citation | Amano, J., Kobayashi, K., and Oshima, M. (2001) Comparative study of
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C., Twu, Y. C., Chou, M. L., Reid, M. E., Gray, A. R., Moulds, J. M. Chang, C. Y., and Lin, M. (2003) The molecular genetics of the human I locus and molecular background explain the partial association of the adult i phenotype with congenital cataracts. Blood 101, 2081-2088 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/36389 | - |
| dc.description.abstract | 癌化發生的過程常會伴隨著細胞表面的醣質結構發生改變，這類的醣結構稱
為tumor-associated antigens；其中最著名的例子為sialyl Lewis a（sLea）及sialyl Lewis x （ sLex ） 在大腸直腸癌中的角色。Lewis 抗原建構於重複的 N-acetyllactosamine（Gal-GlcNAc，LacNAc）單元所形成的poly-LacNAc 結構之 上。這些poly-LacNAc 結構可分為type 1 及type 2 chain，及直鏈型的i 或具支鏈 的I 抗原結構。已知i 及I 抗原的表現會隨發育以及癌症發生過程而變化。然而， 造成sLea 及sLex 在大腸直腸癌細胞中增加的分子機制，以及I 結構在其中扮演 的角色截至目前為止仍不十分明確。本研究主要分為兩大部分，第一部份為建立 腸壁細胞Caco-2 的分化模型；並檢視與Lewis 及I 醣抗原相關基因的表現，以 期能了解Lewis 抗原在癌細胞中表現增加的分子機制及I 醣基因可能的角色。第 二部分為利用此Caco-2 細胞以及K562 erythroleukemia 細胞的分化模型，來研究 I 醣抗原基因表現的調控機制。 在建立的Caco-2 細胞分化模型中，我們檢視參與type 1 chain 合成的 β3GalT5，參與H 抗原合成的FUT1、FUT2，參與Lewis 抗原的FUT3、FUT6、 FUT7 以及I 基因，在細胞分化前後的表現。我們發現在Caco-2 分化後，H type 1 抗原的增加是由於更上游的β3GalT5 基因表現增加所導致。此外，Caco-2 細胞分 化後，造成FUT3 及FUT6 的大量表現，然而我們卻並未觀察到相對應的Lewis 抗原表現量的變化，其中的原因値得進一步的研究。 由於參與I 基因轉錄的IGnTC 在Caco-2 細胞分化後有十分顯著的負調控， 於是建立了IGnTC 正向調控的K562 細胞分化模型，以利研究IGnTC 的調控。 研究結果發現，IGnTC 在兩種細胞模型中的表現，是藉由不同的DNA 區塊來調 控。在Caco-2 細胞中IGnTC 的可能調控區為5’ -2572~-1066 的區域，而K562 細胞，則可能落於5’ -318~-251 約67 bp 的區域內。此段DNA 序列包含Oct-2.1、 Sp1 和C/EBPα轉錄因子的可能結合序列。 | zh_TW |
| dc.description.abstract | Changes of structure and expression profile of cell surface carbohydrates are often
observed in the tumorigenesis. For example, sialyl Lewis a (sLea) and sialyl Lewis x (sLex) epitopes, well known cancer-associated antigens, increased in colorectal cancerous tissues. Lewis antigens have been known building on a poly-LacNAc (Galβ1-3/4GlcNAcβ-, LacNAc) backbone structure, which can be divided into type 1 (Galβ1-3GlcNAcβ-) and type 2 (Galβ1-4GlcNAcβ-) chains. Linear poly-LacNAc structure called i antigen, whereas the branched one called I antigen. The expression pattern of Ii antigens is developmentally regulated and altered during oncogenesis. The molecular mechanism, which leads to cancer-associated expression of sialyl Lewis x/a and the roles of the branched I-antigen structure and I gene in the formation of multimeric Lewis antigen structure have not been well understood. The main study divided into two parts, the first one is establishment of enterocytic differentiation model in Caco-2 cell, and profiling the expression of Lewis- and I-related genes. The second is regarding the regulatory mechanism of I gene in Caco-2 and K562 differentiation models. We have profiled the expression of β3GalT5, FUT1, FUT2, FUT3, FUT6, FUT7, and I genes before and after the differentiation of Caco-2 cells. We observed that the increment of H type 1 antigens after Caco-2 cell differentiated is due to the enhanced expression of upstream β3GalT5 gene. FUT3 and FUT6 genes are highly expressed at transcriptional level after Caco-2 cell differentiated, but the amount of Lewis antigens is not increased. Further investigation is worth doing. As significant down-regulation of IGnTC gene in differentiated Caco-2 cell, we established another K562 cell differentiation model, which IGnTC was up-regulated, to study the regulation mechanism of IGnTC gene. However, the regulatory elements of IGnTC gene in these two cell models are quite different. The possible regulatory element appears to locate on 5’ -2572~-1066 region in Caco-2 cells, whereas in K562 cells the regulatory element of IGnTC gene appears to locate on 5’ -318~-251 region. The sequence of the -318~-251 region contains the potential binding sites for transcription factors Oct-2.1, Sp1 and C/EBPα. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T07:59:09Z (GMT). No. of bitstreams: 1 ntu-94-R92b46014-1.pdf: 749674 bytes, checksum: 67c49129701ab3af6ac3d8243771ca22 (MD5) Previous issue date: 2005 | en |
| dc.description.tableofcontents | 目錄
縮寫表…………………………………………………………………………….….1 摘要…………………………………………………………………………….…..…3 英文摘要（Abstract）………………………………………………………..….4 緒論 一、Lewis 及I 醣抗原與癌化及細胞分化之關係………………………………..5 二、H type 1、H type 2、Lewis 及I 醣抗原之合成路徑以及參與的相關基因…6 1. β3GalT5……………………………………………………………………..7 2. FUT1（H）及FUT2（Se）………………………………………………..8 3. FUT3（Lewis）……………………………………………………………..8 4. FUT6………………………………………………………….………….…9 5. FUT7………………………………………………………………………10 6. IGnT……………………………………………………………………….10 三、Caco-2 及K562 細胞分化模型……………………………………………..11 四、研究構想與目的……………………………………………………………..12 實驗材料與方法 實驗材料…………………………………………………………………………..13 實驗方法………………………………………………………………………......15 一、細胞培養………………………………………………………………….…..15 二、流式細胞儀之分析（Flow Cytometry）………………………………………16 三、半定量反轉錄PCR 分析（Semi-quantitative Reverse Transcription-PCR Analysis）……………………………………………………………….…17 四、5’- and 3’-Rapid Amplification of cDNA End（5’- & 3’-RACE）…………17 五、報導質體之構築（Construction of Reporter Plasmids）……………………19 六、Luciferase-Reporter Assay……………………………………………………19 結果 一、Caco-2 細胞分化模型的建立…………………………………………….….20 二、合成H type 1、H type 2、Lewis 以及I 醣抗原的相關基因，在Caco-2 細胞 分化模型的表現情形……………………………………………………..20 1. 合成H type 1 及H type 2 的相關基因…………………………………..21 2. 合成Lewis 抗原的相關基因……………………………………………..22 3. 合成I 醣抗原的IGnT 基因……………………………………………….24 三、IGnTC 基因調控之研究……………………………………………………..25 1. 建立K562 細胞分化模型………………………………………………...25 2. IGnT 基因在Caco-2 細胞的5’轉錄起始位置及3’未轉譯區域…………25 3. IGnTC 基因在Caco-2 細胞與K562 細胞中轉錄活性變化的比較……..26 4. IGnTC 基因在Caco-2 細胞之可能調控區域…………………………….27 5. IGnTC 基因在K562 細胞之可能調控區域………………………………28 討論………………………………………………………………………………….30 參考文獻…………………………………………………………………………...50 表一半定量RT-PCR 所使用的primers 序列……………………………………35 圖一H type 1 和H type 2 抗原在Caco-2 細胞分化前後之表現情形…………..36 圖二合成H type 1 和H type 2 的相關基因，其transcripts 在Caco-2 細胞分化前後的表現情形………………………………………..……..37 圖三合成Lewis 抗原的相關基因，其transcripts 在Caco-2 細胞分化 前後的表現情形………………………..………………………………..38 圖四Lewis 抗原在Caco-2 細胞分化前後的表現情形……………………….…39 圖五IGnT transcripts 以及I 抗原在Caco-2 細胞分化前後的表現情形……..…40 圖六IGnTC transcripts 以及I 抗原在K562 細胞分化前後的表現情形……..…42 圖七IGnT 基因的5’轉錄起始位置與3’未轉譯區域…………………………..43 圖八用於Luciferase-reporter assay 的各種constructs…………………………..44 圖九IGnTC基因在Caco-2 細胞與K562細胞中轉錄活性變化的比較………..45 圖十IGnTC 基因5’區的不同長度片段，在未分化Caco-2 細胞中之 轉錄活性……..…………………………………………………………..46 圖十一IGnTC 基因5’區的不同長度片段，在K562 細胞之轉錄活性…………..48 圖十二IGnTC 5’ -537~-252 的區域，可能包含的轉錄調控區……………………49 附圖一H type 1、H type 2、Lewis 及I 醣抗原的結構及合成路徑..………………59 附圖二從i 抗原（直鏈型的poly-LacNAc 結構）形成I 抗原（支鏈型的poly-LacNAc 結構）的詳細步驟………………………………………………………..60 附圖三人類I locus 示意圖以及表現的三種IGnT 基因結構……………………..61 附圖四pGL3-basic 報導載體……………………………………………………….62 | |
| dc.language.iso | zh-TW | |
| dc.title | 以細胞分化模型研究Lewis及I醣抗原相關基因
的表現與調控 | zh_TW |
| dc.title | Studies of the Expression Profiles and Regulatory Mechanisms of the Lewis- and I-Antigen Related Genes
in Cell Differentiation Models | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 93-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 朱善德,曾婉芳 | |
| dc.subject.keyword | 醣抗原,細胞分化模型, | zh_TW |
| dc.subject.keyword | Lewis antigen,I antigen,Caco-2, | en |
| dc.relation.page | 62 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2005-07-22 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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