Molecular study of human uterine endometrial cell differentiation for clarifying implantation failure

一般认为，妊娠建立和维持过程中的细胞事件是通过子宫内特异性类固醇调节基因的表达介导的。这些类固醇调节基因的鉴定对于清楚地了解控制子宫内膜生长和分化的分子和细胞过程至关重要。在本研究中，我们试图鉴定与脱个体化相关的基因，然后研究这些基因编码的分子在基质细胞分化和着床过程中的作用。利用减法杂交技术，构建了富含黄体酮依赖基因的cDNA文库。通过分析从这个缺失的cDNA文库中得到的大约2000个克隆，我们发现了12个不同的基因，它们的表达在黄体酮的作用下上调。对3个克隆的动力学分析表明，缺失的cDNA文库可能包含与黄体酮诱导脱个体化的多个步骤有关的基因。其中一个克隆与人类组织金属蛋白酶3抑制剂（TIMP-3）相同，这是最近发现的人类TIMP家族成员。孕酮对人子宫内膜基质细胞培养6天后，timp - 3mrna表达有明显的诱导作用。另外2个基因在黄体酮刺激后2 ~ 6小时诱导；其中一个被发现是组织转谷氨酰胺酶II型（TGase），另一个是未报道的基因。原位杂交显示timp - 3mrna在人子宫内膜前体细胞间质细胞和蜕膜细胞中的表达。由于MMP及其特异性抑制剂TIMP在组织重塑过程中的作用已在多种组织中得到证实，TIMP-3可能对胎儿滋养细胞在胎母界面的侵袭控制起重要作用。另一方面，组织TGase在脱个体化过程中发挥着重要作用，特异的反义寡核苷酸抑制剂抑制该酶的活性，可抑制子宫内膜基质细胞的体外分化。免疫组化研究提示TGase在人子宫内膜向囊胚着床分化过程中的重要作用，对未报道基因（UK10）的Northern blotting检测显示，在反复IVF-ET治疗诊断为着床失败的患者中，该基因的表达显著降低。少

It is generally believed that the cellular events during the establishment and maintenance of pregnancy are mediated through the expression of specific steroid-regulated genes in the uterus. The identification of these steroid-regulated genes is crucial for a clear understanding of the molecular and cellular processes that control uterine endometrial growth and differentiation during pregnancy. In the present study, we have tried to identify the genes involved in decidualization, and then investigated the role of the molecules coded by the genes in stromal cell differentiation and implantation process. By employing subtractive hybridization, we have constructed cDNA library enriched with progesterone dependent genes. While analyzing about 2,000 clones derived from this subtracted cDNA library, we have identified different 12 genes whose expression were up-regulated with progesterone. Kinetic analysis of 3 clones showed that this subtracted cDNA library may contain genes involved in var … More ious step of progesterone-induced decidualization. One of these clones was identical to the human tissue inhibitor of metalloproteinase-3 (TIMP-3), a recently identified member of the human TIMP family. Induction of TIMP-3 mRNA expression was evident when human endometrial stromal cells were cultured with progesterone for 6 days. Other 2 genes were induced as early as 2-6 hours after progesterone stimulation ; one of them was revealed to be tissue transglutaminase type II (TGase) and the other was an unreported gene. In situ hybridization revealed the expression of TIMP-3 mRNA in predecidualized stromal cells and decidual cells of human endometria. Because the role of MMP and their specific inhibitors, TIMP,in the process of tissue remodeling has been demonstrated in various tissues, TIMP-3 may be important for the controlled invasion of fetal trophoblasts at the feto-maternal interface. On the other hand tissue TGase was important in the process of decidualization since the inhibition of the enzyme activity by specific inhibitor of antisense oligonucleotide, the in vitro differentiation of endometrial stromal cells was inhibited. Immunohistochemical studies suggested the importance of TGase in human endometrial differentiation toward blastocyst implantation, and Northern blotting for the unreported gene (UK10) showed that the gene expression was significantly decreased in patients of implantation failure diagnosed by repeated IVF-ET treatment. Less