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

一般认为，妊娠的建立和维持过程中的细胞事件是通过子宫中特定的类固醇调节基因的表达来调节的。识别这些类固醇调节基因对于清楚地了解控制妊娠期间子宫内膜生长和分化的分子和细胞过程是至关重要的。在本研究中，我们试图找出与蜕膜化相关的基因，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。利用消减杂交技术，构建了孕激素依赖基因丰富的cDNA文库。通过对从该消减文库中获得的约2000个克隆的分析，我们鉴定了不同的12个基因，这些基因的表达被孕酮上调。对3个克隆的动力学分析表明，该消减文库可能含有与var…相关的基因孕酮诱导蜕膜形成的IOU数越多。其中一个克隆与人类金属蛋白酶组织抑制因子-3(TIMP-3)完全相同，后者是最近发现的人类TIMP家族成员。人子宫内膜间质细胞与孕酮共同培养6d后，TIMP-3mRNA的表达明显增强。另有2个基因在孕酮刺激后2~6h被诱导，其中一个基因被证实为组织转谷氨酰胺酶II(TGase)，另一个基因为未见报道的基因。原位杂交显示TIMP-3mRNA在人子宫内膜蜕膜前基质细胞和蜕膜细胞中均有表达。由于基质金属蛋白酶及其特异性抑制物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可能在控制胎儿滋养层细胞在母胎界面的侵袭中起重要作用。另一方面，组织TGase在蜕膜形成过程中起重要作用，反义寡核苷酸特异性抑制剂抑制TGase活性，抑制子宫内膜间质细胞的体外分化。免疫组织化学研究表明，TGase在人类子宫内膜向胚泡着床方向分化过程中具有重要作用，对该未报道基因(UK10)进行Northern印迹分析表明，在反复IVF-ET治疗后发现着床失败的患者中，该基因的表达水平显著降低。较少

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