TRANSPORT GENE EXPRESSION AND DRUG ACCUMULATION IN MILK

牛奶中的转运基因表达和药物积累

• 批准号: 6286342
• 负责人: Patrick J McNamara
• 金额: $ 27.32万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6286342
• 关键词: active transport; cell line; cimetidine; excretion; female; gene expression; glucocorticoids; human middle age (35-64); human milk; human tissue; hydantoins; insulin; lactation; mammary epithelium; membrane transport proteins; molecular cloning; northern blottings; polymerase chain reaction; prolactin; radiotracer; tissue /cell culture; transfection; western blottings; women's health; young adult human (21-34)

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.

描述（逐字摘自申请人摘要）：新生儿暴露于 通过牛奶传播多种药物和环境污染物。虽然大多数药物都会获得 通过扩散获得牛奶，我们发现积累 呋喃妥因和西咪替丁比扩散预测多很多倍 模型。这些数据与主动传输过程一致。然而， 负责将这些药物主动转运到牛奶中的蛋白质是 未知，因为对药物转运这一表述知之甚少 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 (SLC22) 的成员和 其他阳离子/阴离子转运蛋白基因存在于乳腺组织中（MRP、OATP、 OCT-1 和 OCT-3)。本研究的目的是系统地评估 ABCB、ABCC、SLC21 和 SLC22 转运蛋白基因家族成员的作用 药物在母乳中的积累。一种基于RT-PCR的定量方法 实时荧光将检验阳离子/阴离子的假设 负责其他组织中药物转运的转运基因， 在乳腺上皮细胞中表达。细胞培养研究将测试 假设永生化的人类乳腺上皮细胞也表达这些 转运蛋白基因，并且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 和蛋白质印迹分析将确认表达和相对丰度 ABCB、ABCC、SLC21 和 SLC22 RNA 和蛋白质水平。功能性摄取和 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积累中表达的转运蛋白 和呋喃妥因，以及模型底物（例如 PAH、TEA）。运输车 发现在哺乳期乳腺细胞中高表达的基因将被克隆 RT-PCR方法并转染至低背景表达的细胞系中 对于这些基因。几种模型底物的摄取和流出，包括 西咪替丁和呋喃妥因将在转染的细胞系中进行测试。 乳腺上皮载体蛋白及其基因的鉴定 控制其监管的因素是这一目标的最终、长期目标 研究计划。