Cellular Zn depletion by metal ion chelators (TPEN, DTPA and chelex resin) and its application to osteoblastic MC3T3-E1 cells.
Cellular Zn depletion by metal ion chelators (TPEN, DTPA and chelex resin) and its application to osteoblastic MC3T3-E1 cells.
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DOI：
10.4162/nrp.2007.1.1.29
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发表时间：
2007
影响因子：
2.4
通讯作者：
Kwun IS
中科院分区：
文献类型：
--
作者：
Cho YE;Lomeda RA;Ryu SH;Lee JH;Beattie JH;Kwun IS
涉及金属离子螯合剂的微量矿物质研究已用于调查某些细胞系的基因和蛋白质表达的反应，但少数研究真正关注这些金属离子螯合剂如何影响重要微量矿物质（如锌、锰、铁和铜）的可用性。本研究的目的是研究在 FBS 中使用螯合树脂并添加 N,N,N',N'-四-(2-吡啶甲基)乙二胺（TPEN，膜渗透性螯合剂）和 处理介质中的二乙烯三胺五乙酸（DTPA，膜不可渗透的螯合剂）。采用原子吸收分光光度计或电感耦合等离子体分光光度计测试了细胞培养基和锌处理培养基的制备成分中Zn、Mn、Fe和Cu的含量。此外，还测量了细胞锌消耗（例如螯合或 TPEN 治疗）时骨相关基因（ALP、Runx2、PTH-R、ProCOL I、OPN 和 OC）的表达。结果表明，在胎牛血清中使用螯合树脂会显着降低可用的 Zn (p<0.05)（39.4 ± 1.5 µM vs 0.61 ± 10.15 µM）和 Mn (p<0.05)（0.74 ± 0.01 µM vs 0.12 ± 0.04 µM）。然而，螯合胎牛血清并没有改变胎牛血清中铁和铜的水平。使用 TPEN 和 DTPA 作为 Zn 螯合剂，处理介质中 Zn 的最终浓度（0、3、6、9、12 µM）没有表现出显着差异，除了添加更高的 15 µM ZnCl2 后，DTPA 螯合处理介质中 Zn 水平显着增加。结果表明，两种螯合剂在 Zn- 和 Zn+ 之间的 5 个骨相关基因的表达模式相同，并且与 chelex 处理的实验相比，TPEN 处理的实验显示出较低的骨相关基因表达，这可能意味着 TPEN 是比 chelex 树脂更强的螯合剂。这项研究表明，与 DTPA 或 chelex 树脂相比，TPEN 是更强的螯合剂，并且 TPEN 和 chelex 树脂产生的细胞锌消耗足以用于细胞锌消耗研究。
Trace mineral studies involving metal ion chelators have been conducted in investigating the response of gene and protein expressions of certain cell lines but a few had really focused on how these metal ion chelators could affect the availability of important trace minerals such as Zn, Mn, Fe and Cu. The aim of the present study was to investigate the availability of Zn for the treatment of MC3T3-E1 osteoblast-like cells and the availability of some trace minerals in the cell culture media components after using chelexing resin in the FBS and the addition of N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN, membrane-permeable chelator) and diethylenetriaminepentaacetic acid (DTPA, membrane-impermeable chelator) in the treatment medium. Components for the preparation of cell culture medium and Zn-treated medium have been tested for Zn, Mn, Fe and Cu contents by atomic absorption spectrophotometer or inductively coupled plasma spectrophotometer. Also, the expression of bone-related genes (ALP, Runx2, PTH-R, ProCOL I, OPN and OC) was measured on the cellular Zn depletion such as chelexing or TPEN treatment. Results have shown that using the chelexing resin in FBS would significantly decrease the available Zn (p<0.05) (39.4 ± 1.5 µM vs 0.61 ± 10.15 µM) and Mn (p<0.05) (0.74 ± 0.01 µM vs 0.12 ± 0.04 µM). However, levels of Fe and Cu in FBS were not changed by chelexing FBS. The use of TPEN and DTPA as Zn-chelators did not show significant difference on the final concentration of Zn in the treatment medium (0, 3, 6, 9, 12 µM) except for in the addition of higher 15 µM ZnCl2 which showed a significant increase of Zn level in DTPA-chelated treatment medium. Results have shown that both chelators gave the same pattern for the expression of the five bone-related genes between Zn- and Zn+, and TPEN-treated experiments, compared to chelex-treated experiment, showed lower bone-related gene expression, which may imply that TPEN would be a stronger chelator than chelex resin. This study showed that TPEN would be a stronger chelator compared to DTPA or chelex resin and TPEN and chelex resin exerted cellular zinc depletion to be enough for cell study for Zn depletion.