Progesterone Withdrawal/Tight Junction Closure--Pregnant

乳腺上皮向大量乳汁分泌的II期转变是由妊娠晚期静止的腔上皮细胞转化为活跃的乳汁分泌细胞的精确级联事件引起的。这种转变是由持续存在高血浆催乳素和正常糖皮质激素时孕酮下降触发的。哺乳期过渡的一个关键事件是紧密连接的闭合，建立起一个物理屏障，将乳腺内腔与间隙空间功能性分离。我们的目标是确定孕酮戒断和紧密连接闭合之间的机制联系。(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)我们将使用蛋白质组学（2D凝胶电泳/质谱法）检查闭合过程中发生的紧密连接蛋白的变化，以确定相关蛋白质。(3)将检查候选调节基因和蛋白质的空间和时间分布，以确定它们的表达方式是否与调节紧密连接闭合的作用一致。(4)关于适当表达的蛋白质的功能假设将在体内进行测试，适当时利用腺病毒载体和转基因和敲除动物的导管内注射。这些实验将提供深入了解紧密连接调节的机制，应该找到广泛的生物适用性。此外，更好地了解产后早期紧密连接闭合的机制和调节将对预防和治疗母乳喂养问题产生重要的临床意义，母乳喂养问题对儿童健康的影响日益得到认可。

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.