原始态水牛胚胎干细胞的培养及Wnt/β-catenin通路对其自我更新作用的初步研究

Embryonic stem cells（ESCs）currently are considering as two pluripotent states: naïve and primed. It is widely believed that most of embryonic stem cells (ESCs) from livestock are primed state. In recent years, the studies have indicated that Wnt/β-Catenin signal pathway plays an important role in activating the naïve embryonic stem cells. In this project, firstly, the possibility of obtaining the naïve buffalo ESCs through cells reprogramming and combining with the technology of embryonic stem cells are investigated. Secondly, the expression pattern of target genes mRNA and protein of the Wnt/β-Catenin signal pathway in the buffalo pluripotent ESCs are examined. Synchronously, the effects of activators (CHIR99021 and BIO) and inhibitor (IWR-1-endo) of Wnt/β-Catenin signaling pathway on the culture efficiency, as well as the expression of target genes and the proteins of Wnt/β-Catenin signaling pathway in buffalo pluripotent ESCs are also detected. This will help to clarify the function of Wnt/β-Catenin signaling pathway in the self-renewal of buffalo ES cells, enrich and consummate the regulation network of self-renewal in buffalo ES cells, so that to optimize the culture system of buffalo ES cells, and successfully establish bonafide ES cells line in buffalo.

目前建立的胚胎干细胞（embryonic stem cells, ESCs）被分为原始态（Naïve ）和 始发态（primed）两种状态，现在普遍认为来源大家畜的ES细胞大都处于primed状态。近年来的研究发现Wnt/β-Catenin信号通路是激活原始态ES细胞重要标志路径。本项目拟利用重编程方法结合胚胎干细胞技术探讨获取原始态多能性水牛ES细胞的方法；同时研究多能性水牛ES细胞中Wnt/β-catenin信号通路相关成分在mRNA和蛋白水平上的表达规律，并探讨Wnt/β-catenin通路激活剂（CHIR99021和BIO）及抑制剂（IWR-1-endo）对多能性水牛ES细胞培养效果和通路相关基因及蛋白表达的影响，从而阐明Wnt/β-catenin通路在ES细胞自我更新的调控作用。这将有助于丰富和完善水牛ES细胞自我更新的调控网络，对建立稳定的水牛ES细胞培养体系具有重要意义。

本项目主要围绕原始态水牛胚胎干细胞（ES）体外分离培养的相关技术环节以及Wnt/β-catenin信号通路对其自我更新作用进行了初步研究。首先优化了水牛卵母细胞和胚胎体外培养体系，为水牛ES细胞的分离培养提供质量更好的胚胎，初步弄清了褪黑素通过与细胞膜 G 蛋白偶联受体MT1和MT2 结合，抑制了cAMP合成，提高cGMP含量，从而促进卵母细胞成熟及早期胚胎发育；同时还发现在水牛卵母细胞体外成熟培养液中添加适宜浓度的BDNF（10ng/ml）有利于水牛卵母细胞体外成熟及发育潜能的获得。其次，研究了原始态水牛胚胎干细胞分离培养的方法，通过探讨水牛囊胚发育阶段对原始态/始发态胚胎干细胞分离培养效果的影响，确定了水牛囊胚多能性基因表达主要集中在桑椹胚和早期囊胚，经2i处理早期胚胎后能够促进水牛多能性ESCs原代克隆的形成，并利于维持原代克隆原始态多能性基因的表达。第三，探讨了Wnt/β-catenin信号通路对多能性水牛胚胎干细胞多能性维持和自我更新的作用，通过添加Wnt/β-catenin通路上GSK3特异性抑制剂BIO和CHIR99021研究其对水牛多能性ES细胞培养效果的影响，发现GSK3抑制剂（CHIR99021和BIO）对水牛多能性ES细胞增殖和多潜能性的维持具有促进作用，初步揭示了Wnt/β-catenin信号通路对水牛多能性胚胎干细胞自我更新起着重要的作用。另外，还探讨了碱性成纤维细胞生长因子对促进水牛类胚胎生殖干细胞的作用，明确了外源性bFGF在水牛PGCs重编程为EG-like细胞过程中发挥了关键作用，为优化水牛ES细胞的培养提供了参考依据。