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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 劉?睿(Je-Ruei Liu) | |
dc.contributor.author | Yu-Hsuan Wang | en |
dc.contributor.author | 王毓瑄 | zh_TW |
dc.date.accessioned | 2021-06-17T06:38:11Z | - |
dc.date.available | 2023-08-19 | |
dc.date.copyright | 2018-08-19 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-08-15 | |
dc.identifier.citation | 參考文獻
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Tanaka, Y., Y. Taki, T. Sakane, T. Nadai, H. Sezaki, and S. Yamashita. 1995. Characterization of drug transport through tight-junctional pathway in Caco-2 monolayer:comparison with isolated rat jejunum and colon. Pharm Res. 12:523-528. Ude, V. C., D. M. Brown, L. Viale, N. Kanase, V. Stone, and H. J. Johnston. 2017. Impact of copper oxide nanomaterials on differentiated and undifferentiated Caco-2 intestinal epithelial cells;assessment of cytotoxicity, barrier integrity, cytokine production and nanomaterial penetration. Part Fibre Toxicol. 14:31. Vergauwen, H. 2015. The IPEC-J2 cell line. The Impact of Food Bioactives on Health. p. 125-134. Veum, T., M. Carlson, C. Wu, D. Bollinger, and M. Ellersieck. 2004. Copper proteinate in weanling pig diets for enhancing growth performance and reducing fecal copper excretion compared with copper sulfate. J Anim Sci. 82:1062-1070. Wang, J., X. Zhu , Y. Guo, Z. Wang, B. Zhao, Y. Yin, and G. Liu. 2016. Influence of dietary copper on serum growth-related hormone levels and growth performance of weanling pigs. Biol Trace Elem Res. 172:134-139. Weiss, K. H., J. C. Lozoya, S. Tuma, D. Gotthardt, J. Reichert, R. Ehehalt, W. Stremmel, and J. Füllekrug. 2008. Copper-induced translocation of the Wilson disease protein ATP7B independent of Murr1/COMMD1 and Rab7. Am J Pathol. 173:1783-1794. Zakrzewski, S. S., J. F. Richter, S. M. Krug, B. Jebautzke, I. F. M. Lee, J. Rieger, M. Sachtleben, A. Bondzio, J. D. Schulzke, M. Fromm, and D. Günzel. 2013. Improved cell line IPEC-J2, characterized as a model for porcine jejunal epithelium. PLoS One. 8:e79643. Zetzsche, A., N. Schunter, J. Zentek, and R. Pieper. 2016. Accumulation of copper in the kidney of pigs fed high dietary zinc is due to metallothionein expression with minor effects on genes involved in copper metabolism. J Trace Elem Med Biol. 35:1-6. Zhao, J., A. F. Harper, M. J. Estienne, K. E. Webb Jr., A. P. McElroy, and D. M. Denbow 2007. Growth performance and intestinal morphology responses in early weaned pigs to supplementation of antibiotic-free diets with an organic copper complex and spray-dried plasma protein in sanitary and nonsanitary environments. J Anim Sci. 85:1302-1310. Zhou, W., E. T. Kornegay, M. D. Lindemann, J. W. Swinkels, M. K. Welten, and E. A. Wong. 1994. Stimulation of growth by intravenous injection of copper in weanling pigs. J Anim Sci. 72:2395-2403. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/72369 | - |
dc.description.abstract | 飼料中添加銅具有促進豬隻生長表現的效果,然而,超過90%的銅未受吸收利用而隨糞便排出,蓄積於土壤造成環境汙染。本試驗利用人類結腸癌細胞株(the human colon carcinoma cell line, Caco-2)以及豬小腸上皮細胞株(porcine intestinal epithelial cell line, IPEC-J2)細胞模型模擬銅離子在腸道的吸收,以探討多種有機銅化合物是否具有促進銅吸收之效果。試驗中測試之物質包括:胺基酸lysine與methionine、類黃酮(flavonoid)化合物rutin與quercetin,以及乳鐵蛋白(lactoferrin),另以市售產品Zincpro之Availa-Cu 100與傳統添加的硫酸銅(CuSO4)做為對照組。將細胞培養於透析膜上,測定跨膜電阻(transepithelial electrical resistance, TEER)及其對於螢光染劑的通透量,並利用免疫螢光染色觀察緊密連結蛋白zonula occludens-1(ZO-1),以評估單層細胞膜的完整性。並以掃描式電子顯微鏡(scanning electron microscopy, SEM)確認單層細胞膜之分化形態。待細胞模型建立後,將上述測試物質分別加入含有單層細胞之透析膜上,收集上層及下層之溶液,以感應耦合電漿原子發射質譜儀(inductively coupled plasma mass spectrometry, ICP-MS)進行銅的定量,並分析細胞之銅相關蛋白之基因表現。結果顯示,quercetin和methionine能顯著地促進Caco-2與IPEC-J2對於銅的吸收,而rutin的添加也能促進Caco-2對於銅的吸收。Caco-2在添加quercetin、rutin,以及methionine後,ATP7A的基因表現顯著較控制組高,推論這三種化合物能促進銅離子吸收,並將銅離子由ATP7A輸送至透析膜下層,但Ctr1與DMT1的基因表現並無顯著變化。IPEC-J2在添加quercetin、rutin,以及methionine後,Ctr1的基因表現顯著降低,顯示Ctr1可能參與銅離子的吸收,methionine的添加顯著提高IPEC-J2的DMT1基因表現。根據研究結果顯示,Caco-2與IPEC-J2在添加篩選物質後對於銅離子的吸收機制並不相同,quercetin和methionine可能具有促進銅吸收之應用潛力。 | zh_TW |
dc.description.abstract | High level of copper sulfate (CuSO4) is typically supplemented to swine diets as a growth promoter since numerous studies have demonstrated that Cu supplementation at pharmacological levels improves growth performance in swine. However, because of the low absorption of Cu, over 90% of dietary Cu is excreted into the manure and results in serious accumulation of Cu in soil. Therefore, it is necessary to develop compounds for improving Cu absorption. It is possible that organically complexed minerals could be absorbed by several pathways, which could be more bioavailable to animals than the inorganic minerals. Accordingly, numerous organic agents can be considered as alternative supplements for enhancement of Cu absorption. The aim of this study is to establish in vitro Cu absorption models to screen the Cu-absorption enhancement compounds, including amino acids (such as lysine and methionine), lactoferrin, and flavonoids (such as rutin and quercetin), and their effects on copper transporter genes expression. Also, commercially available copper-amino acid complex and CuSO4 were used as control groups. The human colon adenocarcinoma cell line (Caco-2) and porcine intestinal epithelial cell line (IPEC-J2) were employed to evaluate the Cu-absorption efficiency. The tight junction integrity of the cell monolayer is assessed by the measurement of transepithelial electrical resistance (TEER) and the membrane integrity marker, lucifer yellow. The models are further characterized structurally by scanning electron microscopy, showing the differentiation phase of the intestinal epithelial cell. As for the Cu absorption assays, the results show that quercetin, rutin, and methionine enhanced Cu absorption and ATP7A mRNA expression in Caco-2 cell model. Quercetin and methionine also facilitated copper absorption in IPEC-J2 cell model. Though quercetin, rutin, and methionine significantly reduced Ctr1 mRNA expression, and methionine significantly enhanced DMT1 mRNA expression in IPEC-J2 cell. It is suggested that Ctr1 and DMT1 mediated Cu absorption of IPEC-J2 cell. In conclusion, Caco-2 and IPEC-J2 show different regulatory mechanisms of Cu to the supplements. Quercetin and methionine might be alternative supplements for enhancement of Cu absorption. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T06:38:11Z (GMT). No. of bitstreams: 1 ntu-107-R05626022-1.pdf: 2402991 bytes, checksum: e01bf3e3ae96f5b24ef77952384de773 (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 目錄
口試委員會審定書 # 誌謝 i 中文摘要 ii Abstract iii 目錄 v 圖目錄 viii 表目錄 x 第一章 文獻探討 1 一、飼料銅對於豬的影響 1 (一)飼料添加物之定義 1 (二)生理功能 1 (三)餵飼添加量 1 (四)對於豬隻生長表現之影響 2 二、銅的吸收與代謝 4 (一)銅相關運輸蛋白 4 (二)影響銅吸收的因子 4 三、不同形式之銅添加 7 四、腸道細胞體外吸收模型 7 五、研究目標 8 第二章 材料與方法 11 一、實驗架構 11 二、細胞株活化、繼代及冷凍保存 12 (一)細胞活化 12 (二)繼代培養 12 (三)冷凍保存 13 三、腸道上皮細胞模型之建立 13 (一)銅吸收實驗用細胞準備 13 (二)單層細胞膜完整性測定 13 (三)單層細胞膜形態分析 14 四、硫酸銅與具促進銅吸收的潛力物質之細胞毒性測定 17 五、銅吸收實驗 18 (一)促進銅吸收之潛力物質對於銅跨膜轉運及細胞內含量之影響 18 (二)回收率測定 19 六、銅運轉蛋白之基因表現 19 七、統計方法 23 第三章 結果與討論 24 一、腸道上皮細胞模型之建立 24 (一)單層細胞膜完整性測定 24 (二)單層細胞膜形態分析 24 二、硫酸銅與具促進銅吸收的潛力物質之細胞毒性測定 33 三、銅吸收實驗 39 (一)促進銅吸收之潛力物質對於銅跨膜轉運及細胞內含量之影響 39 (二)回收率測定 40 四、銅運轉蛋白之基因表現 53 第四章 結論 73 參考文獻 74 圖目錄 圖1-1、銅於腸細胞的吸收 6 圖2-1、實驗架構 11 圖2-2、腸道上皮細胞模型 16 圖3-1、培養天數對於Caco-2單層細胞膜TEER值的影響 25 圖3-2、培養天數對於IPEC-J2單層細胞膜TEER值的影響 26 圖3-3、培養天數對於lucifer yellow通透Caco-2單層細胞膜的影響 27 圖3-4、培養天數對於lucifer yellow通透IPEC-J2單層細胞膜的影響 28 圖3-5、Caco-2單層細胞膜的緊密連結蛋白ZO-1之表現 29 圖3-6、IPEC-J2單層細胞膜的緊密連結蛋白ZO-1 之表現 30 圖3-7、以掃描式電子顯微鏡觀察Caco-2單層細胞膜的表面形態 31 圖3-8、以掃描式電子顯微鏡觀察IPEC-J2單層細胞膜的表面形態 32 圖3-9、以MTT法測試CuSO4對細胞存活率之影響 34 圖3-10、篩選之化合物與硫酸銅共培養對Caco-2細胞存活率之影響 35 圖3-11、篩選之化合物與硫酸銅共培養對IPEC-J2細胞存活率之影響 36 圖3-12、各處理組對於Caco-2單層細胞膜TEER值的影響 37 圖3-13、各處理組對於IPEC-J2單層細胞膜TEER值的影響 38 圖3-14、Caco-2細胞於銅吸收實驗中各處理組之銅離子濃度 42 圖3-15、IPEC-J2細胞於銅吸收實驗中各處理組之銅離子濃度 43 圖3-16、評估之化合物對於通透Caco-2細胞之銅離子累積量的影響 44 圖3-17、評估之化合物對於Caco-2表觀滲透係數的影響 45 圖3-18、評估之化合物對於Caco-2細胞內銅含量的影響 46 圖3-19、評估之化合物對於通透IPEC-J2細胞之銅離子累積量的影響 47 圖3-20、評估之化合物對於IPEC-J2表觀滲透係數的影響 49 圖3-21、評估之化合物對於IPEC-J2細胞內銅含量的影響 50 圖3-22、Caco-2細胞銅吸收實驗中銅離子的回收百分比 51 圖3-23、IPEC-J2細胞銅吸收實驗中銅離子的回收百分比 52 圖3-24、Quercetin對Caco-2細胞之mRNA表現量的影響 55 圖3-25、Rutin對Caco-2細胞之mRNA表現量的影響 57 圖3-26、Methionine對Caco-2細胞之mRNA表現量的影響 60 圖3-27、Availa-Cu對Caco-2細胞之mRNA表現量的影響 62 圖3-28、Quercetin對IPEC-J2細胞之mRNA表現量的影響 64 圖3-29、Rutin對IPEC-J2細胞之mRNA表現量的影響 65 圖3-30、Lysine對IPEC-J2細胞之mRNA表現量的影響 67 圖3-31、Methionine對IPEC-J2細胞之mRNA表現量的影響 69 圖3-32、Availa-Cu對IPEC-J2細胞之mRNA表現量的影響 71 表目錄 表1-1、豬隻對於銅的營養需求及建議添加量 3 表1-2、國家標準之豬配合飼料含銅最高限量 9 表1-3、Availa-Cu 100之配方 10 表2-1、Caco-2引子列表 21 表2-2、IPEC-J2引子列表 22 | |
dc.language.iso | zh-TW | |
dc.title | 利用腸道細胞模型篩選具有促進銅吸收的物質 | zh_TW |
dc.title | Screening of the Potential Compounds for Enhancing Copper Absorption by Intestinal Epithelial Cell Models | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄭光成,劉?德,李滋泰 | |
dc.subject.keyword | 銅吸收,硫酸銅,有機銅化合物,人類結腸癌細胞株,豬小腸上皮細胞株, | zh_TW |
dc.subject.keyword | Copper absorption,Copper sulfate,Organically complexed copper,Caco-2 cell line,IPEC-J2 cell line, | en |
dc.relation.page | 80 | |
dc.identifier.doi | 10.6342/NTU201803535 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-08-16 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 動物科學技術學研究所 | zh_TW |
顯示於系所單位: | 動物科學技術學系 |
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