Influence of zinc deficiency on the mRNA expression of zinc transporters in adult rats
Influence of zinc deficiency on the mRNA expression of zinc transporters in adult rats
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DOI：
10.1016/s0946-672x(03)80005-6
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发表时间：
2003-01-01
影响因子：
3.5
通讯作者：
Windisch, W
中科院分区：
文献类型：
--
作者：
Pfaffl, MW;Windisch, W
锌在细胞中的积累是通过四种锌转运蛋白(如四种锌转运蛋白，如四种锌转运蛋白(ZnT1-4)、二价阳离子转运蛋白1(DCT1))和主要与金属硫蛋白(MT)结合的储存过程的内流和外流过程的总和。以成年大鼠为动物模型，研究缺锌对其基因表达水平的影响。缺锌组大鼠摄入量为8g/d，含纯植酸锌强化2g/g，对照组(n=7)为58g/g。分别于缺锌第1、2、4、7、11、16、22、29天处死3只动物(n=24)。锌缺乏明显表现为血浆锌、血浆碱性磷酸酶活性降低和锌从组织库(主要是骨骼)中的严重动员，而摄食量和体重没有受到影响。取代表锌吸收(空肠、结肠)、锌储存和利用(肌肉、肝脏)和锌排泄(肾脏)的组织。结肠中总核糖核酸含量增加(p=0.003)，肝脏中总核糖核酸含量呈下降趋势(p=0.086)。缺锌对肌肉组织和肾脏组织总RNA含量无明显影响。建立了实时反转录(RT)聚合酶链式反应(PCR)检测方法，并建立了基于GAPDH的相对定量方法。检测可以相对准确地定量信使核糖核酸分子，具有足够高的灵敏度和重复性。所有已知的锌转运蛋白亚型均在组织中发现。最近在大鼠组织中发现并测序了一组新的基因序列。对于被分析的组织，表达模式和对缺锌的反应是特定的。表达结果表明，一些转运蛋白是结构性表达的，而另一些转运蛋白在负责锌稳态的组织中受到高度调控。表达水平变化最明显的是结肠，因此可以推测这是一个高度锌敏感的组织。MT在所有组织中均有表达下调，在肝脏(p<；0.001)和结肠(p=0.002)中大量下调，在空肠和肾脏中也有下调趋势。该基因与细胞内锌状态平行，是锌缺乏的有效候选基因。锌组和锌组大鼠结肠组织中的基因表达显著上调(p=0.032和p=0.026)，而锌组的空肠组织中的基因表达则呈下降趋势(p=0.098)。本研究首次在恒定平台上对非生长期成年大鼠模型的基因表达调控进行了比较研究，并对所有已知锌转运蛋白的表达进行了全面的定量分析，因此可以进行直接比较。
The accumulation of zinc in the cell is a sum of influx and efflux processes via transporter proteins, Like the four Zn transporters (ZnT1-4), the divalent cation transporter 1 (DCT1) and of storage processes mainly bound to metallothionein (MT). To study the effect of Zn deficiency on mRNA expression Levels, adult rats were used as an animal model. Food intake was restricted to 8 g/day containing 2 mug Zn/g fortified with pure phytate in Zn deficiency rats and 58 mug Zn/g in controls (n = 7). At day 1, 2, 4, 7, 11, 16, 22, and 29 of Zn deficiency, 3 animals were sacrificed, respectively (n = 24). Zn deficiency was evident from reduced plasma Zn, plasma alkaline phosphatase activity and severe mobilization of Zn from tissue stores (mainly skeleton), while food intake and body weight remained unaffected. Tissues representing Zn absorption (jejunum, colon), Zn storage and utilization (muscle, Liver), and Zn excretion (kidney) were retrieved. Total RNA contents increased in colon (p = 0.003) and trend to decrease in Liver (p = 0.086). Zn deficiency was without effect on tissue total RNA concentrations in muscle tissue and kidney. Real-time reverse transcription (RT) polymerase chain reaction (PCR) assays were developed and a relative quantification on the basis of GAPDH was applied. Assays allowed a relative and accurate quantification of mRNA molecules with a sufficiently high sensitivity and repeatability. ALL known Zn transporter subtypes were found in the tissues. ZnT3 was newly elucidated and sequenced in rat tissues. Expression patterns and reactions to Zn deficiency were specific for the tissue analysed. Expression results imply that some transporters are expressed constitutively, whereas others are highly regulated in tissues responsible for Zn homeostasis. The most distinct changes of expression Levels were shown in colon which can therefore be postulated as a highly Zn sensitive tissue. MT was down-regulated in all tissues, massively in Liver (p < 0.001) and in colon (p = 0.002) and in tendency also in the jejunum and kidney. In parallel with intracellular Zn status it is a potent candidate gene for Zn deficiency. ZnT1 and ZnT2 showed a significant up-regulation of mRNA expression in colon (p = 0.032 and p = 0.026) and for ZnT2 a trend of down regulation in jejunum (p = 0.098). This study provides the first comparative view of regulation of gene expression and fully quantitative expression analysis of all known Zn transporters in a non growing adult rat model on a constant platform and therefore allows a direct comparison.