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利用牛子宫胎盘组织的细胞外基质（ECM）和滋养细胞与子宫内膜相互作用对子宫内膜功能的调控机制，建立模拟牛胚胎着床的体外研究工具。尿素缓冲液和脱氧胆酸钠能有效地提取子宫胎盘组织中的ECM，并能促进牛子宫内膜成纤维细胞的生长。在脱氧胆酸钠提取的ECM上，成纤维细胞生长良好，形成坚固的适应性强的细胞片。该薄片可作为产生子宫内膜样多细胞结构的可利用基质。微阵列和原位杂交分析表明，胎盘乳原、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对着床起关键作用，ECM降解相关酶；膜型I-MMP和细胞外基质金属蛋白酶诱导剂。EMMPRIN参与了牛子宫内膜重塑和着床。PRP-1特异性结合基底膜组分，IV型胶原。该分子可能是牛早期胚胎与子宫内膜上皮粘连着床的关键因素。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，滋养层细胞能产生一些特异性分子，有效刺激成纤维细胞的生长。总的来说，牛子宫-胎盘ECM基质可能保留了在体外使用细胞和基质成分再生多细胞结构的有用特征，并且本研究中ECM降解酶和粘附分子的信息可能加速体外子宫内膜模型的建立，然而，建立它们需要更复杂的安排。

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.