Studies on the genes and molecules involved in human endometrial cell proliferation and differentiation

对于成功植入人体，包括基质细胞蜕膜转化在内的子宫内膜的精确定时变化被认为是非常重要的。虽然脱个体化主要受黄体分泌的黄体酮控制，但最近的研究表明，多种其他因素在脱个体化的调控中起着局部介质的作用。孕激素作用于子宫内膜间质细胞的过程中可能存在一些第二信使分子。在这项研究中，我们建立了一个体外人去个体化模型。在黄体酮存在下培养的人子宫内膜基质细胞发生形态分化并产生蜕膜蛋白，如PRL。利用这种体外脱个体化模型，我们试图确定与脱个体化有关的基因。从增殖性子宫内膜组织中分离人子宫内膜基质细胞，利用poly (A)+ rna构建cDNA文库。采用cDNA减法、表达法、多射线法和差异显示法对黄体酮诱导子宫内膜间质细胞分化相关基因进行了鉴定。结果鉴定出组织转谷氨酰胺酶（Tgase）、TIMP-3、CD63、IL-15和UK10未报道的基因等多个基因。组织Tgase基因在孕激素培养6小时后诱导，未报道的UK10基因在1小时内诱导，孕激素培养7-10天后，CD63下调，IL-15上调。组织Tgase是Tgase家族的一员，它催化多肽中谷氨酰胺残基的g-羧酰胺基团与多肽中赖氨酸残基的伯胺或e-氨基之间的钙依赖性酰基转移反应。组织酶被报道存在于各种器官和组织中。组织Tgase的生物学功能尚不完全清楚，但有人认为该酶在调节细胞生长和分化、细胞膜钙依赖性交联、细胞程序性死亡以及细胞因子的激活和二聚化等方面发挥作用。由于在组织Tgase基因中加入特定的底物竞争对手或反义寡脱氧核苷酸，可以抑制孕激素诱导的脱个体化，因此表明组织Tgase在脱个体化中的重要作用。虽然子宫内膜组织Tgase的底物尚不清楚，但该分子对于黄体酮诱导的间质细胞分化是必不可少的。对于UK10基因，我们发现该基因在中期秘书期子宫内膜中表达，在着床失败患者中表达降低。孕酮可显著提高子宫内膜基质细胞IL-15 mRNA水平及IL-15的产生。提示IL-15的产生刺激了子宫NK细胞的诱导，是成功建立妊娠的关键事件。此外，我们还首次明确了孕酮调控基因20a-羟基类固醇脱氢酶。少

For successful implantation in humans, precisely timed changes in the endometrium including the decidual transformation of stromal cells are believed to be very important. Although decidualization is primarily under the control of progesterone secreted by corpus luteum, recent studies have demonstrated that various other factors act as local mediators in the regulation of decidualization. And there should exist some second messenger molecules in progesterone action on endometrial stromal cells. In this study, we have developed an in vitro model of human decidualizaiton. Human endometrial stromal cells cultured in the presence of progesterone undergo morphological differentiation and produce decidual proteins such as PRL. With this model of in vitro decidualization, we tried to identify the genes involved in decidualization. Human endometrial stromal cells from proliferative endometrial tissues were isolated and cDNA libraries were made from poly (A)+RNAs. cDNA subtraction, expression a … More rray, and differential display methods were used to identify genes involved in endometrial stromal cell differentiation induced by progesterone. As a results, several genes including tissue transglutaminase (Tgase), TIMP-3, CD63, IL-15, and unreported gene of UK10 were identified. Tissue Tgase gene was induce after 6 hours culture with progesterone and the unreported gene, UK10, was induced within 1 hour culture, CD63 was down regulated and IL-15 was unregulated by progesterone after 7-10 days culture with progesterone. The enzyme tissue Tgase is a member of the Tgase family that catalyzes calcium-dependent acyl transfer reaction between g-carboxamide group of the glutamine residues in peptides and either primary amines or e-amino groups of the lysine residues in peptides. Tissue Tgase is reported in various organs and tissues. The biological function of tissue Tgase is not completely understood, but it is suggested that the enzyme has roles in regulation of cell growth and differentiation, in calcium-dependent cross linking of cell membrane, in the programmed cell death, and in the activation and dimerization of cytokines. The important role of tissue Tgase in decidualization was suggested, since the addition of a specific substrate competitor, or the antisense oligodeoxynucleotide to tissue Tgase gene inhibited progesterone-induced decidualizaiton. Although the substrate(s) of tissue Tgase in endometrium for is unknown, the molecule(s) should be indispensable for stromal cell differentiation induced by progesterone. As to UK10 gene, we found the gene was expressed in mid-secretary phase endometrium, and its expression was decreased in patients of implantation failure. IL-15 mRNA levels as well as IL-15 production by endometrial stromal cells was apparently increased by progesterone. And it was suggested that IL-15 production stimulates the induction of uterine NK cells and is a key event for the successful pregnancy establishment. In addition, we have clarified a gene for progesterone regulation, 20a-hydroxysteroid dehydrogenase for the first time. Less