TRANSPORT GENE EXPRESSION AND DRUG ACCUMULATION IN MILK

描述(逐字摘自申请者摘要)：新生儿暴露在 各种药物和环境污染物通过牛奶。虽然大多数药物都有收益 通过扩散获得乳汁，我们发现积累的 呋喃妥因和西咪替丁比扩散法预测的要高出许多倍 模特们。这些数据与正在进行的传输过程一致。然而， 负责将这些药物主动转运到牛奶中的蛋白质是 不知道，因为对药物转运的表达知之甚少 乳房上皮细胞中的蛋白质。来自我们的初步研究 实验室表明，溶质载体家族22(SLC22)和 其他阳离子/阴离子转运蛋白基因存在于乳腺组织中(MRP，OATP， 10月1日和10月3日)。本研究的目标是系统地评估 ABCB、ABCC、SLC21和SLC22转运蛋白家族成员在细胞周期中的作用 药物在母乳中的积聚。一种基于DNA序列的定量RT-PCR方法 实时荧光将检验阳离子/阴离子的假说 负责药物在其他组织中运输的转运蛋白基因， 在乳腺上皮细胞中表达。细胞培养研究将测试 永生化的人类乳腺上皮细胞也表达这些 转运蛋白基因，这些基因的一个子集被上调 催乳激素(即催乳素、胰岛素和糖皮质激素)。北方 Western印迹分析将证实表达和相对丰度 ABCB、ABCC、SLC21和SLC22的RNA和蛋白水平。功能性摄取和 对这些永生化细胞的外排研究将证实这些细胞的作用 放射性标记西咪替丁特异性蓄积中的转运蛋白表达 和呋喃妥因，以及模型底物(例如，多环芳烃、茶)。传送器 已发现在哺乳期乳腺细胞中高表达的基因将被克隆 RT-PCR方法并转入低背景表达的细胞系 这些基因。几种模型底物的吸收和外流，包括 西咪替丁和呋喃妥因将在转基因细胞系中进行测试。 乳腺上皮细胞载体蛋白及其基因的鉴定 控制其监管的因素是最终的、长期的目标。 研究计划。

DESCRIPTION (Verbatim from the Applicant's Abstract): Neonates are exposed to a variety of drugs and environmental contaminants via milk. While most drugs gain access to milk by diffusion, we have found that the accumulation of nitrofurantoin and cimetidine is many-fold greater than predicted by diffusion models. These data are consistent with an active transport process. However, the proteins responsible for the active transport of these drugs into milk are not known, since little is known regarding the expression drug transport proteins in lactating mammary epithelial cells. Preliminary studies from our laboratory indicate that members of the Solute Carrier Family 22 (SLC22) and other cation/anion transporter genes are present in mammary tissue (MRP, OATP, OCT-1 and OCT-3). The goal of this research is to systematically evaluate the role of members of the ABCB, ABCC, SLC21 and SLC22 transporter gene families in the accumulation of drugs into human milk. A quantitative RT-PCR method based on real-time fluorescence will test the hypothesis that cation/anion transporter genes which are responsible for drug transport in other tissues, are expressed in mammary epithelial cells. Cell culture studies will test the hypothesis that immortalized human mammary epithelial cells also express these transporter genes and that a subset of these genes are up-regulated by lactogenic hormones (i.e., prolactin, insulin and glucocorticoids). Northern and Western blot analysis will confirm the expression and relative abundance of ABCB, ABCC, SLC21 and SLC22 RNA and protein levels. Functional uptake and efflux studies in these immortalized cells will substantiate the role of these expressed transporters in the specific accumulation of radiolabeled cimetidine and nitrofurantoin, as well as model substrates (e.g., PAH, TEA). Transporter genes found to be highly expressed in lactating mammary cells will be cloned by RT-PCR methods and transfected into cell lines with low background expression for these genes. The uptake and efflux of several model substrates, including cimetidine and nitrofurantoin, will be tested in the transfected cell lines. The identification of the mammary epithelial carrier proteins, their genes, and factors that control their regulation are the ultimate, long-term goals of this research program.