Study of the embryo-derived factors in endometrial stromal cells

子宫内膜基质细胞胚胎源性因子的研究

基本信息

批准号：
09470357
负责人：
NODA Yoichi
金额：
$ 5.5万
依托单位：
Shiga University of Medical Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Progress in understanding the mechanism of implantation has been hampered by the complexity of cellular interactions between embryos and endometrial stromal cells (ESCs). Although in vitro system of implantation may contribute to understanding such complex biological phenomena in vivo, it is ethically impossible to use human embryos in these experiments. Therefore, the establishment of in vitro model of mouse implantation is mandatory to understand the mechanism of implantation. As a first step, we tried to establish in vitro model of decidualization. Mouse ESCs were collected and incubated in DMEM supplemented with 10% FCS, estradiol (E2, 0.1nM) and progesterone (P, 100nM).ESCs cultured with EP transformed into large and decidua-like cells and produced decidual protein. Ultrastructurally, these cells became to have abundant rough endoplasmic reticulum during the transformation. These findings show that mouse ESCs in culture respond to ovarian steroids and showed morphological and func … More tional changes similar to decidualization in vivo. Thus, this culture system may serve as an in vitro model of mouse decidualization.As a second step, development of embryos and invasion of the trophoblast into ESCs were studied using this in vitro. model of decidualization. ESCs isolated from 4 week old mice were cultured for 9 days in DMEM supplemented with E2 (0.1 nM) and P (100 nM). The blastocysts isolated from 4 day pregnant mice were co-cultured with ESCs with or without E2/P, and the blastocysts were also cultured without ESCs as control (single culture). After 12-day culture, the blastocysts development and their interaction with ESCs were examined under light microscopy and transmission electron microscopy. The results were as follows : 1) blastocysts in the single culture were degenerated by the 12th day of culture regardless of E2/P addition, while the blastocysts co-cultured with ESCs survived without degeneration ; 2) in the co-culture, the area of growing embryos significantly increased in E2/P containing medium, compared with those without E2/P ; 3) the invasion of trophoblasts into ESCs was apparent in medium without E2/P, while it was inhibited in medium with E2/P. ; 4)ESCs were transformed into decidua-like cells in spite of culture without E2/P when they were co-cultured with blastocysts. These results suggest that decidual cells enhance the embryonic development and regulate the trophoblastic invasion, while embryos may produce some decidualization-stimulating factor(s), thus indicating the presence of a mutual interaction between decidualized ESCs and embryos. Less

胚胎和子宫内膜基质细胞（ESC）之间细胞相互作用的复杂性阻碍了理解植入机制的进展，尽管体外植入系统可能有助于理解体内这种复杂的生物现象，但在伦理上不可能使用人类。因此，建立小鼠胚胎的体外植入模型对于了解植入机制是必要的。作为第一步，我们尝试建立小鼠胚胎干细胞的蜕膜化模型。 DMEM补充有10％FCS、雌二醇(E2，0.1nM)和黄体酮(P，100nM)。用EP培养的ESC转化为大的蜕膜样细胞并产生蜕膜蛋白。从超微结构上看，这些细胞变得具有丰富的粗面内质网。这些发现表明，培养中的小鼠 ESC 对卵巢类固醇有反应，并表现出形态和功能……更多因此，该培养系统可以作为小鼠蜕膜化的体外模型。第二步，使用该体外蜕膜化模型研究胚胎的发育和滋养层向ESC的侵袭。从 4 周龄小鼠中分离的 ESC 在补充有 E2 (0.1 nM) 和 P (100 nM) 的 DMEM 中培养 9 天。妊娠4天的小鼠与含有或不含E2/P的ESC共培养，并且囊胚也以不含ESC的方式培养作为对照（单一培养）。培养12天后，在光下检查囊胚的发育及其与ESC的相互作用。显微镜检查和透射电镜检查结果如下： 1）无论培养第12天，单一培养物中的囊胚均发生退化。添加E2/P，而与ESC共培养的囊胚存活且没有变性；2）共培养中，含有E2/P的培养基中生长的胚胎面积较不含E2/P的培养基显着增加； )在不含E2/P的培养基中滋养层细胞对ESC的侵袭明显，而在含有E2/P的培养基中则受到抑制；4)尽管培养，ESC仍转化为蜕膜样细胞。当它们与囊胚共培养时，没有E2/P，这些结果表明蜕膜细胞增强胚胎发育并调节滋养层侵袭，而胚胎可能产生一些蜕膜化刺激因子，从而表明存在相互作用。蜕膜化 ESC 和胚胎之间的差异较小。