Progesterone Withdrawal/Tight Junction Closure--Pregnant

乳腺上皮向大量乳汁分泌的第二阶段转变是由妊娠晚期静止的管腔上皮细胞转变为活跃的乳汁分泌细胞的一系列精确的事件所致。这种转变是由持续存在高血浆催乳素和正常糖皮质激素的情况下孕酮的下降触发的。过渡到哺乳的一个关键事件是紧密连接的关闭，建立起一种物理屏障，提供乳腺管腔与间质空间的功能性分离。我们的目标是确定黄体酮停用和紧密连接关闭之间的机制联系。(1)我们将使用微阵列分析来确定伴随这一转变的调控基因的变化。(2)我们将利用蛋白质组学(2D凝胶电泳法/质谱仪)检测封闭过程中紧密连接蛋白的变化，以确定相关蛋白质。(3)将检测候选调控基因和蛋白的时空分布，以确定它们的表达方式是否与紧密连接封闭的调节作用一致。(4)利用导管内注射腺病毒载体以及适当的转基因和敲除动物，将在体内测试关于适当表达的蛋白质的功能假说。这些实验将提供对紧密连接调节机制的洞察，这些机制应该会找到广泛的生物学适用性。此外，更好地了解产后早期紧密连接关闭的机制和调节，将对预防和治疗母乳喂养问题具有重要的临床意义，这些问题对儿童健康的影响日益得到承认。

The stage II transition of the mammary epithelium to copious milk secretion results from a precise cascade of events that transform the quiescent luminal epithelial cells of late pregnancy into active milk secreting cells. This transition is triggered by a fall in progesterone in the continued presence of high plasma prolactin and normal glucocorticoid. A critical event in the transition to lactation is the closure of tight junctions, erecting a physical barrier that provides functional separation of the lumen of the mammary gland from the interstitial space. Our goal is to determine the mechanistic linkage between progesterone withdrawal and tight junction closure. (1) We will determine changes in regulatory genes that accompany this transition using microarray analysis. (2) We will examine changes in tight junction proteins that occur during closure using proteomics (2D gel electrophoresis/mass spectrometry) to identify the relevant proteins.(3) The spatial and temporal distribution of candidate regulatory genes and proteins will be examined to determine whether they are expressed in a manner consistent with a role in regulation of tight junction closure. (4) Functional hypotheses about proteins that are expressed appropriately will be tested in vivo, utilizing intraductal injection of adenoviral vectors and transgenic and knock-out animals as appropriate. These experiments will provide insight into the mechanisms of tight junction regulation that should find broad biological applicability. In addition a better understanding of the mechanisms and regulation of tight junction closure during the early post-partum period will have significant clinical implications for the prevention and treatment of breast-feeding problems whose impact on child health is increasingly recognized.