利用腸道細胞模型篩選具有促進銅吸收的物質

Screening of the Potential Compounds for Enhancing Copper Absorption by Intestinal Epithelial Cell Models

王毓瑄(Yu-Hsuan Wang)

指導教授 ： 劉嚞睿

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201803535

摘要

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。

關鍵字

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

並列摘要

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.

並列關鍵字

Copper absorption ； Copper sulfate ； Organically complexed copper ； Caco-2 cell line ； IPEC-J2 cell line

參考文獻

參考文獻

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