牛体细胞核移植（SCNT）类滋养层干细胞基因表达和分化形成双核滋养层细胞能力的研究

Somatic cell nuclear transfer (SCNT) has been employed to produce clones of a wide variety of mammalian species since the first successful use of differentiated donor cells was reported. However, the success of cloning by nuclear transfer has not come without complication. Fewer than 6% of the reconstructed bovine blastocysts develop into viable offspring. From the time of embryo transfer (day 8) to day 90 of gestation, 70% of SCNT pregnancies are lost. A major problem associated with the mortality of these reconstructed embryos is poor placental development, which has been reported earlier in gestation and during the critical time of early post implantation. The trophoblast binucleate/giant cells (TGC), which makes up approximately 20% of the trophoblast population, and these cells produce pregnancy-related proteins, such as pregnancy-associated glycoproteins (PAGs), placental lactogen (PL), and prolactin-related proteins. Researches indicted that TGC development in SCNT embryos is altered. Given the morphological variation and consequent dysfunction of TGC in placentas from SCNT embryos, it is of considerable interest to explore the expression of factors involved in bovine trophoblast differentiation during the early stages of placental formation. The trophoblast stem cells derived from trophoectderm is the best tool to explore the trophoblast differentiation in vitro. Using a system include two inhibitors (PD0325901 and CHIR99021), we have established bovine trophoblast stem-like cells, which exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers, such as OCT4, NANOG, SOX2, SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81, and trophoblast lineage markers such as CDX2, as determined by both immunofluorescence and RT-PCR. Additionally, these cells generated dome-like structures, formed teratomas when injected into NOD-SCID mice, and differentiated into placenta trophoblast cells in vitro. Based on the preliminary work, this project will further study of the difference in gene expression,methylation status of key maker genens and differentiation potentials between AI、IVF and SCNT derived trophoblast stem-like cells, explore the formation of placenta dysplasia and provide strategies to improve successful rate of SCNT embryos.

自从世界首例体细胞克隆绵羊多莉出生以来，体细胞核移植技术已经被广泛应用于各种哺乳动物。牛是克隆动物中最成功的一个种类，尽管如此，这一技术的应用仍然存在流产率高及胎盘、胎儿发育异常等问题。牛克隆胚胎在植入前和植入后早期的丢失率达到70%左右，其中双核滋养层巨细胞发育异常可能在胎盘发育异常中起重要作用。利用含有两个小分子抑制剂(PD0325901和CHIR99021)的培养体系，我们从IVF和SCNT胚胎成功获得可以在外稳定培养和传代的牛类滋养层干细胞系，二者在形态和特性上没有显著差异，但是经基因表达谱芯片分析，二者在表达基因有明显差异。本项目将在前期工作基础上，深入研究AI、IVF和SCNT类滋养层干细胞之间在基因表达、甲基化和分化形成包括双核滋养层巨细胞在内的成熟滋养层的差异，为探索SCNT胚胎胎盘发育异常和预防提供新的思路和策略。

滋养层干细胞是胎盘细胞的前体细胞，是体外研究胎盘形成的有效工具。利用2i (PD0325901和CHIR99021)培养系统，建立人工授精（AI）、体外受精（IVF）和核移植（SCNT）囊胚来源的牛类滋养层干细胞，实时定量PCR和免疫荧光染色检测分析，发现牛IVF和SCNT类滋养层干细胞在形态、干性基因表达和分化特征等方面没有显著差异。通过基因表达谱芯片检测和基因聚类分析，发现IVF和SCNT来源的牛类胚胎干细胞的基因表达图谱存在差异，SCNT高表达的基因共有140类，其中74类差异显著，主要集中在线粒体功能和脂类合成方面，在SCNT低表达的基因有 132类，其中57类差异显著，主要集中在线粒体功能和细胞迁移生长相关基因方面。利用亚硫酸氢盐测序法对4种干细胞关键基因的甲基化分析，结果显示，与IVF类滋养层干细胞（BTS1）相比，SCNT类滋养层干细胞（BTSA）中CDX2、OCT4、NANOG和SOX2基因的启动子区的甲基化程度都有所下降。CDX2的甲基化从1.67%降低到1.13%，OCT4的甲基化从52.23%降低到39.57%，NANOG的甲基化从82.30%降低到64.01%，SOX2的甲基化从3.00%降低到0.70%。通过对牛类滋养层干细胞进行体外分化，发现IVF和SCNT双核滋养层巨细胞形成的区别，这些结果为深入研究SCNT胎盘发育异常和提高SCNT胚胎的出生率提供了新思路。