TEAD家族蛋白（TEAD4）基因对牛早期胚胎发育及胚胎干细胞多能性和分化调控的研究

Embryonic stem cells (ESCs) are a kind of pluripotent stem cells that can be isolated from inner cell mass (ICM) of blastocysts, maintained in an artificially induced self-renewal state in vitro, and keep highly similar character to ICM cells. Compared to ICM of rodent and primate, bovine ICM had different pattern of gene expression. Thus, bovine ESCs (bESCs) have not been successfully derived yet after numerous attempts based on the experience from rodents or human. All previous bESCs cannot be continuously passaged in vitro and their partial pluripotency gradually lost during long-term cultivation. It significantly restricted to generate genetically modified cattle by stem cells technology. Previous studies confirmed that several trophectoderm-specific genes were detectable in bovine ICM. However, it is unclear that effect of these genes on bovine embryonic development and pluripotent maintenance of embryonic stem cells. In this study, effect of TEAD4, one of trophectoderm-specific genes for specifying the trophectoderm lineage at the beginning of mammalian development, on bovine pre-implantation embryonic development, and the mechanism that it regulates pluripotent maintenance and differentiation of bESCs will be investigated. By this study, it will contribute to establish a novel in vitro culture system for long-term culturing bESCs, and will be cornerstone for using bovine stem cell technology in generating genetically modified cattle.

胚胎干细胞是从内细胞团中分离，可体外长期增殖，并保持类似内细胞团特性的多能干细胞。与啮齿类和灵长类动物不同，牛囊胚内细胞团在基因表达模式等方面存在明显特异性。因而，至今模仿啮齿类和灵长类动物干细胞培养体系并不能完全维持牛胚胎干细胞的多能性。目前培养的牛胚胎干细胞不能长期稳定增殖，且不具有完整的多能性，严重制约干细胞技术在家畜育种领域中的应用。研究显示，牛囊胚内细胞团表达多个滋养外胚层基因。然而，这些基因对于牛早期胚胎发育和干细胞多能性的调控机制还不清楚。本项目在前期研究的基础上，进一步探讨滋养外胚层发育关键调控因子，TEAD家族蛋白TEAD4在牛早期胚胎发育中的调控作用，进而研究它对牛胚胎干细胞多能性维持、分化及建系的影响。阐明内细胞表达的滋养外胚层基因对牛干细胞多能性调控的分子机制，为建立适合牛胚胎干细胞长期培养、稳定维持多能性的培养体系以及将牛干细胞技术应用于基因修饰牛的制备奠定基础。

胚胎干细胞是一类具有分化潜能的细胞，它能够分化形成构成机体的所有细胞类型。然而，目前只有小鼠、大鼠和人的胚胎干细胞被成功分离、培养。牛的胚胎干细胞虽经历20多年的研究仍未获得。目前认为，对牛早期胚胎发育机制研究的匮乏是导致至今无法获得牛胚胎干细胞的关键因素。本研究从探讨早期胚胎内细胞团和滋养层的发育调控着手，重点探讨发育调控基因TEAD4对于牛早期胚胎发育和干细胞多能性建立的影响。研究显示，牛TEAD4基因在8-细胞期激活表达，并在桑葚胚期迅速上调，在囊胚期有所下降。在牛早期胚胎中敲减TEAD4基因，虽然囊胚率显著下降，但囊胚腔依然能够形成，当分离内细胞团用于干细胞培养时，TEAD4敲减的干细胞不能长期培养。因此，牛TEAD4在调控早期胚胎发育和干细胞多能性方面有其物种的特异性。.另一方面，本研究还将牛LIF（bLIF）cDNA构建到真核表达载体pSIN-EF2-IRES-Pur中，获得稳定表达bLIF的cos7细胞系，并获得了条件培养液，证实，bLIF条件培养液可以长期维持小鼠ESCs细胞多能性，并且利用该条件培养液结合2i小分子化合物以及bFGF，可以获得了可长期培养牛iPSCs细胞的培养体系。.此外，由于TEAD4基因在肌肉卫星细胞中表达，本研究也探讨了TEAD4对于牛肌肉卫星细胞分化的影响。本研究证实，TEAD4的敲减不影响肌肉干细胞向肌管、神经、骨和胰腺的分化，但是显著影响其分化为脂肪。与对照组相比，TEAD4敲减肌肉干细胞获得的脂肪细胞脂滴含量少、相关成脂分化的基因表达收到影响。