Abstract：
Early embryonic mortality resulting from insufficient interaction between the embryo and the uterus leads to the failure of pregnancy in livestock animals. Thus, it is imperative to comprehend the multifaceted process of implantation at molecular levels, which requires synchronized feto-maternal interaction. The in-vitro models serve as valuable tools to investigate the specific stages of implantation. The present study was undertaken to develop a simple method to isolate and culture the primary buffalo endometrial epithelial cells (pBuEECs), followed by proteome profiling of the proliferating cells. Collagenase I was used to separate uterine epithelial cells (UECs) from the ipsilateral uterine horn, and then the cells were separated using a cell strainer. After being seeded on culture plates, UECs developed colonies with characteristic epithelial shape and expressed important markers such as cytokeratin 18 (KRT18), progesterone receptor (PGR), β-estrogen receptor (ESR1), and leukemia inhibitory factor (LIF), which were confirmed by PCR. The purity of epithelial cells was assessed using cytokeratin 18 immunostaining, which indicated approximately 99% purity in cultured cells. The proteome profiling of pBuEECs via high-throughput tandem mass spectrometry (MS), identified a total of 3383 proteins. Bioinformatics analysis revealed enrichment in various biological processes, including cellular processes, metabolic processes, biological regulation, localization, signaling, and developmental processes. Moreover, the KEGG pathway analysis highlighted associations with the ribosome, proteosome, oxidative phosphorylation, spliceosome, and cytoskeleton regulation pathways. In conclusion, these well characterized cells offer valuable in-vitro model to enhance the understanding of implantation and uterine pathophysiology in livestock animals, particularly buffaloes.
摘要：
由于胚胎和子宫之间的相互作用不足而导致的早期胚胎死亡导致家畜动物妊娠失败。因此，必须在分子水平上理解植入的多方面过程，这需要胎儿-母体的同步互动。体外模型是研究植入具体阶段的有价值的工具。本研究旨在开发一种简单的方法来分离和培养原代水牛子宫内膜上皮细胞（pBuEEC）。然后是增殖细胞的蛋白质组分析。胶原酶I用于从同侧子宫角分离子宫上皮细胞（UEC），然后用细胞过滤器分离细胞。接种在培养板上后，UECs形成了具有特征性上皮形状的集落，并表达了重要的标志物，如细胞角蛋白18（KRT18），孕激素受体（PGR），β-雌激素受体（ESR1），和白血病抑制因子（LIF），经PCR证实。使用细胞角蛋白18免疫染色评估上皮细胞的纯度，这表明培养细胞的纯度约为99%。通过高通量串联质谱（MS）对pBuEECs进行蛋白质组分析，共鉴定出3383种蛋白质。生物信息学分析揭示了在各种生物过程中的富集，包括细胞过程，代谢过程，生物调节，本地化,信令,和发展过程。此外，KEGG通路分析强调了与核糖体的关联，蛋白体,氧化磷酸化，剪接体,和细胞骨架调节途径。总之,这些特征良好的细胞提供了有价值的体外模型，以增强对家畜动物植入和子宫病理生理学的理解，尤其是水牛。
