Copper Transport in Lactation

描述（由申请人提供）：铜被不同种类的细胞（包括转运体和所涉及的传递蛋白）从血浆中吸收的机制的许多方面，以及铜如何（在细胞进入后）找到特定分泌物的方式，仍有待确定。我们已经获得的证据表明，哺乳深刻地改变了新吸收的铜的组织分布，将大部分铜从肝脏和肾脏转移到乳腺，并迅速出现在牛奶和牛奶铜蓝蛋白中。牛奶中铜蓝蛋白中的铜可能比牛奶中其他形式的铜对婴儿更有生物可利用性，这一点尚未得到很好的界定。为了研究乳腺上皮细胞的泌乳作用，建立了一种对泌乳激素反应的极化细胞培养模型。突变的啮齿类动物模型缺乏特定的蛋白质，这些蛋白质可能参与乳腺吸收铜及其输送到发育中的乳汁所需的步骤。长期目标是了解在正常条件下以及妊娠和哺乳期铜的分布和运输是如何调节的。当前的目标是确定在正常情况下和哺乳期，可交换血浆池中的铜是如何进入乳腺（和肝脏）细胞的，以解释在后一种情况下观察到的分布的显著变化（涉及哪些潜在的膜转运蛋白，以及它们的表达和/或处置如何因哺乳期而改变）。目的还在于确定在细胞进入后，铜是如何被输送到牛奶和牛奶酰胺蛋白的；ATOX1、WND和/或MNK在这一过程中以及在调节牛奶中铜含量方面的作用；并进一步确定哺乳动物奶中的铜成分。通过Real Time PCR和免疫分析，分别在mRNA和蛋白质水平上测量潜在转运蛋白的表达；用免疫荧光和共聚焦显微镜进行定位。在哺乳激素、转染和反义寡核苷酸的细胞培养模型中，会诱导表达的改变。将确定全动物模型中特定转运体的自然和敲除突变对铜分布和运输的影响。铜在牛奶和牛奶铜蓝蛋白中的吸收和出现，随后是67Cu/64Cu和免疫沉淀。将测量针对膜转运蛋白的特异性抗体对铜摄取的影响。牛奶分泌物中的铜结合成分将被表征。预计这些研究将大大提高我们对铜如何从血液中分配到细胞的认识，特别是乳腺上皮细胞如何将铜放入可能对婴儿产生特定影响的牛奶成分中。

DESCRIPTION (provided by applicant): Many aspects of the mechanisms by which Cu is taken up from the blood plasma by different kinds of cells (including the transporters and delivery proteins involved), and just how (after cell entry) Cu finds its way to specific secretions, remain to be defined. We have obtained evidence that lactation profoundly alters the tissue distribution of newly absorbed Cu, diverting most of it from the liver and kidney to the mammary gland, where it rapidly appears in the milk and in milk ceruloplasmin. Cu in milk ceruloplasmin may be more bio-available to the infant than the other forms of Cu in the milk, which have not been well defined. A polarized cell culture model responsive to lactational hormones has been developed for studying milk production by mammary epithelial cells. Mutant rodent models lacking specific proteins that could be involved in the steps required for uptake of Cu by mammary gland and its delivery to developing milk are available. The long term objective is to understand how Cu distribution and transport are regulated under normal conditions and in gestation and lactation. The immediate objectives are to determine how Cu from the exchangeable plasma pool enters mammary (and hepatic) cells, under normal conditions and in lactation, to explain the marked changes in distribution observed in the latter condition (what potential membrane transporters are involved, and how their expression and/or disposition is changed by lactation). The objectives are also to determine how, after cell entry, copper is routed to the milk and milk cemloplasmin; the role(s) of ATOX1, WND and/or MNK in this process and in regulating the copper content of the milk; and to further define the copper components of mammalian milk. Expression of potential transporters will be measured at the mRNA and protein levels, by Real Time PCR and immunoassays, respectively; and localization will be followed with immunofluorescence and confocal microscopy. Alterations in expression will be induced in the cell culture model with lactational hormones, transfection, and antisense oligos. Effects of natural and knockout mutations of specific transporters in whole animal models on copper distribution and transport will be determined. Cu uptake and its emergence in milk and milk ceruloplasmin will be followed with 67Cu/64Cu and immunoprecipitation. Effects on Cu uptake of specific antibodies against membrane transporters will be measured. Cu binding components in milk secretions will be characterized. It is expected that the proposed studies will greatly advance our knowledge of how Cu is distributed to cells from the blood and particularly how mammary epithelial cells put Cu into components of the milk that may have specific effects on the infant.