TRANSPORT GENE EXPRESSION AND DRUG ACCUMULATION IN MILK

描述（来自申请人摘要的逐字描述）：新生儿暴露于 各种药物和环境污染物通过牛奶。虽然大多数药物 通过扩散获得牛奶，我们发现， 呋喃妥因和西咪替丁的浓度比扩散预测值高出许多倍 模型这些数据与主动运输过程一致。然而，在这方面， 负责将这些药物主动转运到乳汁中的蛋白质是 未知，因为关于表达药物转运知之甚少 哺乳期乳腺上皮细胞中的蛋白质。我们的初步研究 实验室表明溶质载体家族22（SLC 22）成员， 其它阳离子/阴离子转运蛋白基因存在于乳腺组织中（MRP，OATP， OCT-1和OCT-3）。本研究的目的是系统地评估 ABCB、ABCC、SLC 21和SLC 22转运蛋白基因家族成员在 药物在母乳中的积累。一种定量RT-PCR方法， 实时荧光将测试假设，阳离子/阴离子 负责药物在其他组织中转运的转运蛋白基因， 在乳腺上皮细胞中表达。细胞培养研究将测试 假设永生化人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 蛋白质印迹分析将证实 ABCB、ABCC、SLC 21和SLC 22 RNA和蛋白水平。功能性摄取和 在这些永生化细胞中的外排研究将证实这些 放射性标记西咪替丁特异性蓄积中表达的转运蛋白 和呋喃妥因，以及模型底物（例如，PAH，TEA）。转运蛋白 发现在泌乳乳腺细胞中高度表达的基因将被克隆， RT-PCR方法并转染到具有低背景表达的细胞系中 对于这些基因。几种模型底物的吸收和流出，包括 西咪替丁和呋喃妥因将在转染的细胞系中进行测试。 乳腺上皮细胞载体蛋白及其基因的鉴定， 控制其调节的因素是最终的，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.