Regeneration of cell culture substrata using bovine uterine and placentomal extra-cellular matrix and remodeling of endometrial functions

本研究的目的是探索牛子宫胎盘组织细胞外基质(ECM)基质的使用方法，以及滋养层细胞与子宫内膜在着床前后相互作用对子宫内膜功能的调节机制，以期建立一种模拟牛着床的体外研究工具。尿素缓冲液和脱氧胆酸钠能有效地从子宫胎盘组织中提取细胞外基质，并能刺激牛子宫内膜成纤维细胞的生长。成纤维细胞在脱氧胆酸钠提取的ECM上生长良好，形成了牢固的、适应性强的细胞膜。该片材可以是用于产生子宫内膜样多细胞结构的可利用的基质。基因芯片和原位杂交分析表明，胎盘催乳素、催乳素相关蛋白-1(PRP1)、PRP5、妊娠相关糖蛋白7、组织蛋白酶K、胰岛素生长因子结合蛋白等分子在着床过程中起关键作用，细胞外基质降解相关酶、膜型I-MMPs和细胞外基质金属蛋白酶诱导物在着床过程中起重要作用。EMMPRIN参与了牛子宫内膜的改建和着床。PRP-1与基底膜成分IV型胶原特异性结合。该分子可能是牛早期胚胎与子宫内膜上皮黏附和着床的关键因素。滋养层细胞系(BT-1细胞)与子宫内膜成纤维细胞共培养表明，滋养层细胞产生某些特定的分子来有效地刺激成纤维细胞的生长。总体而言，牛子宫胎盘ECM的底物可能保留了利用细胞和基质成分在体外再生多细胞结构的有用特征，而且本研究中的ECM降解酶和黏附分子的信息可能会加速体外子宫内膜模型的建立，然而，需要更复杂的安排来建立它们。

The aim of this study was to explore the way to use of a new extra-cellular matrix (ECM) substrata from bovine utero-placental tissues and the regulatory mechanisms for endometrial functions regarding the interaction between trophoblast cells and endometrium around implantation for establishing a mimic in vitro research tool of bovine implantation. Urea buffer solution and sodium deoxycholate were effective to extract ECM from utero-placental tissues, and these extracts stimulated the growth of bovine endometrial fibroblasts. Fibroblasts well grew and developed a firm and adaptable cell-sheet on ECM extracted by sodium deoxycholate. The sheet may be an utilizable substratum for generating endometrial-like multi-cellular structure. Microarray and in situ hybridization analysis suggested that some molecules, like placental lactogen, prolactin-related proteins -1 (PRP1), PRP5, pregnancy-associated glycoprotein 7, cathepsin K, insulin growth factor binding protein, took a crucial role for implantation, and ECM degradation related enzymes; membrane type I-MMP and extracellular matrix metalloproteinase inducer. EMMPRIN, were participated in the remodeling of endometrium and implantation in bovine. PRP-1 specifically bound to basement membrane component, type IV collagen. The molecule may be a key factor for adhesion and implantation between early embryo and endometrial epithelium in bovine. Co-culture of trophoblast cell line (BT-1 cell) and endometrial fibroblasts suggested that trohoblast cell produced some specific molecule to stimulate fibroblast cell growth efficiently. Overall the substrata from bovine utero-placental ECM may retain an useful feature for regeneration of multi-cellular structure using cells and matrix components in vitro, and also the information of ECM degradation enzymes and adhesion molecules in this study may accelerate establishing in vitro endometrial models, however, more sophisticated arrangements need for establishing them.