MDR1 IN HUMAN INTESTINE

描述：多药耐药(MDR)基因编码P- 糖蛋白(PGP)是一种多功能的三磷酸腺苷依赖泵， 将各种结构上无关的物质从 减少细胞内细胞毒药在细胞质内的积聚。 Pgp最初是在肿瘤细胞中发现的，但现在已知是 在包括肠腔表面在内的正常细胞中表达 上皮细胞。并不是所有的Pgp都会产生耐药性。校长 研究人员已经在大鼠和人身上表明，只有功能形式， Mdr1a和mdr1分别在小鼠中显著表达 肠子。初步数据还表明，MDR在 肠道是外源生物屏障的一部分。校长 调查人员还发现，有显著的个体间 多药耐药基因在小肠组织中的表达变化。 首席研究员认为，这种异质性有助于 患者间对多药耐药底物的口服清除差异，可能 代表了以前未被认识到的环境风险因素 疾病。要检验的第一个假设是， 多药耐药基因在人肠道中的表达主要是非遗传性的 各种因素。首席调查者根据他的研究假设 初步研究表明mdr1a的mrna和蛋白是高度 在大鼠肠道内可被多种外源物质诱导。以特定的目标 1，首席调查员将确定mdr1 mRNA和mdr1基因的诱导者 处理培养的人肠道的小肠和结肠中的蛋白质 带有一组可疑诱导剂的外植体。在具体目标2中， 在AIM 1中确定的诱导剂将通过测量MDR1在体内进行测试 获取小肠和大肠活检组织中的mRNA和蛋白质 在无诱导剂饮食4天后，内窥镜下形成健康受试者 在用诱导剂进行短期治疗后再次出现。肠道外植体 从每个受试者身上获得的也会暴露在相同的诱导剂中 体外培养。首席研究人员还将测试饮食的影响 健康志愿者饮食对肠道多药耐药基因表达的影响 含有木炭烤牛肉。多药耐药基因mdr1的表达水平 将在研究中获得的小肠和大肠活检中确定 在4天无诱导剂饮食后进入，并在7天后进行测试 节食。首席研究员将与保罗·沃特金斯博士合作 关于西柚汁(类黄酮类)和 十字花科蔬菜(吲哚)。具体目标3将直接评估 肠道多药耐药水平影响吸收的假说 肠道中的药物。首席调查员将首先 检测诱导多药耐药基因表达对白血病细胞阈值的影响 环孢素(CsA)在大鼠离体肠的吸收。 多药耐药蛋白水平和转运功能之间的相关性也将 在大鼠肠道刷状缘小泡中被测定(协作 理查德·莫斯利)。第二，首席调查员将 确定肠道中个体间异质性的贡献 多药耐药对环孢素A、咪达唑仑和17α-阿司匹林口服动力学的影响 健康受试者中的乙炔雌二醇(与保罗·沃特金斯合作)。 在具体目标4中，首席调查员将确定是否 Caco-2细胞对多药耐药诱导剂有反应 特定目标1和2.发现一种反应灵敏的人类细胞系 使首席调查员能够确定基因的特征 未来多药耐药基因表达的差异。现建议本局 从计划的研究中获得的数据将导致新的和可测试的 关于药物生物利用度和环境风险的假设 评估。

DESCRIPTION: The multiple drug resistance (mdr) genes encode P- glycoproteins (Pgp) which function as versatile ATP dependent pumps, transporting a variety of structurally unrelated substances from the cytoplasm to reduce the intracellular accumulation of cytotoxic drugs. Pgps were initially identified in tumor cells but are now known to be expressed in normal cells including the lumenal surface of intestinal epithelia. Not all Pgps confer drug resistance. The principal investigator has shown in rat and man that only the functional forms, mdr1a and mdr1 respectively, are significantly expressed in small intestine. Preliminary data also suggests that mdr functions in the intestine as part of a barrier to xenobiotics. The principal investigator has also found that there is significant interindividual variation in the expression of mdr1 mRNA and protein in the small bowel. The principal investigator believes this heterogeneity contributes to interpatient differences in the oral clearance of mdr substrates and may represent a previously unrecognized risk factor for environmental diseases. The first hypothesis to be tested is that variation in expression of mdr1 in human intestine is largely due to non genetic factors. The principal investigator bases this hypothesis on his preliminary studies that demonstrated mdr1a mRNA and protein are highly inducible in rat intestine by a variety of xenobiotics. In Specific Aim 1, the principal investigator will identify inducers of mdr1 mRNA and protein in small bowel and colon by treating cultured human intestinal explants with a battery of suspected inducers. In Specific Aim 2, the inducers identified in Aim 1 will be tested in vivo by measuring mdr1 mRNA and protein in small and large bowel biopsies obtained endoscopically form healthy subjects after a 4 day inducer free diet and again after short term treatment with the inducer. Intestinal explants obtained from each subject will also be exposed to the same inducer in vitro. The principal investigator will also test the effects of diet on intestinal mdr expression by placing healthy volunteers on diets containing charcoal broiled beef. The level of mdr1 mRNA and protein will be determined in small and large bowel biopsies obtained at study entry, after a 4 day inducer free diet, and after 7 days on the test diet. The principal investigator will collaborate with Dr. Paul Watkins on similar studies involving grapefruit juice (flavonoids) and cruciferous vegetables (indoles). Specific Aim 3 will directly assess the hypothesis that the level of intestinal mdr influences the absorption of drugs in the intestine. The principal investigator will first examine the effect of inducing mdr expression on the threshold for cyclosporine (CSA) absorption in an isolated loop of rat intestine. Correlation between mdr protein levels and transport function will also be determined in rat intestinal brush border vesicles (collaboration with Richard Moseley). Second, the principal investigator will determine the contribution of interindividual heterogeneity in intestinal mdr expression to the oral kinetics of CSA, midazolam, and 17 alpha- ethinyl estradiol in healthy subjects (collaboration with Paul Watkins). In Specific Aim 4, the principal investigator will determine whether CaCo-2 cells are responsive to the mdr inducers identified in the Specific Aims 1 and 2. The discovery of a responsive human cell line would allow the principal investigator to characterize the genetic differences in mdr expression in the future. It is proposed that the data obtained from the planned studies will lead to novel and testable hypotheses regarding the bioavailability of drugs and environmental risk assessment.