家兔胚胎干细胞Naïve多能性维持与调控机制的研究

Pluripotency of embryonic stem cells (ESC) can be divided into two distinct states: naïve and primed. Naïve state presents a robust and potential ability to be germline competent. The previously established rabbit embryonic stem cells (rbESC) exhibited primed pluripotency which are more like moue epiblast stem cells (EpiSC) and human embryonic stem cells (hESC). Studies have indicated that protein kinase C signal pathway (PKC) regulates rodent and human ESC self-renewal. However, the role of PKC has not been defined in other mammals. Our previous study found that the inhibition of PKC signaling supports the derivation of rbESC with the morphology as dome-shaped, appearing to maintain naïve properties, including the expression of naïve transcriptional regulators, such as AP, Oct4, Nanog, Sox2 and Klf4. It implies that these derived rbESC may be at naïve state, and regulated by PKC. We will define whether the pluripotent properties of rbESC are belong to naïve state similar to mESC, then analyze the mechanism of PKC in regulating self-renewal and differentiation, and finally investigate the interaction of PKC with other signaling pathways to maintain rbESC pluripotency and naïve state. This study provides an insight into the naïve pluripotency in rbESC, and the molecular mechanism to establish an authentic rbESC for germline transmission.

多能性胚胎干细胞（ESC）分为Naïve和Primed两种状态。Naïve状态的ESC具备更强的分化潜能与种质传递的能力。在家兔中仅建立了Primed rbESC。啮齿目和人类的研究发现，Naïve ESC的维持与自我更新受到PKC信号通路的调控。我们发现，在抑制GSK3和ERK信号通路的基础上，进一步抑制PKC能够促进家兔ICM形成“Dome-shaped”状态的rbESC，细胞可高表达AP、Oct4、Nanog、Sox2、Klf4等多能性分子。维持rbESC细胞“Dome-shaped”的状态受到PKC抑制物剂量的影响，结果显示rbESC具有潜在的Naïve多能性，且受PKC的调控。本课题将界定“Dome-shaped”状态的rbESC是否具备Naïve的特性，阐明PKC调控rbESC自我更新的分子机制，揭示PKC与其它信号通路对Naïve rbESC的协同机理。

动物与人类的胚胎干细胞（ESC）具有两种多能性状态，原始态（Naïve ）和始发态（Primed）。Naïve态ESC具有更强的发育多能性，具有种质传递能力。目前，在家兔中仅建立了Primed状态的家兔胚胎干细胞，Naïve态家兔胚胎干细胞（Naïve-rbESC）还未被分离到。本项目初步分离了Naïve-rbESC，并鉴定其为Naïve 态。.通过筛选多个信号通路抑制剂，筛选出了分离Naïve-rbESC的培养基（rb3i），分离的Naïve-rbESC传至P5代并保持未分化状态。分析发现，Naïve-rbESC形态与Naïve-mESC细胞集落形态相似，都是呈“Domed”隆起状，AP染色呈阳性。免疫荧光染色显示，细胞株除高表达Oct4，Sox2，Klf4等多能性标志细胞因子，最重要的是该细胞也表达 mESC Naïve态标志蛋白Rex1，Fgf4。结果表明获得的Naïve-rbESC特征与Naïve-mESC非常相似，属于Naïve状态。.rb3i培养基中bFGF和抑制剂PD0325901、CHIR99021、Gӧ6983发挥着重要作用，为探究其机理，我们在rb3i培养体系中分别去除这四种因子，发现缺少以上任何一种因子都无法成功分离Naïve-rbESC，表明bFGF和抑制剂PD0325901、CHIR99021、Gӧ6983在捕获rbESC Naïve态中起相互协同作用。PKC信号（PKCi）在捕获Naïve态中起重要作用，我们以小鼠Naïve态mESC为模型，比较了2iL和PKCi条件捕获的Naïve态转录组特征，差异表达基因达3543个；2iL-mESC以高表达TBX3、Klf4、Nanog为特征，而PKCi-mESC以高表达Lin28a、Myc为特征，Naïve与Primed态的标志基因表达模式也表现出较大差异，结果表明PKCi与经典2iL条件维持的Naïve态具有不同特征。.本研究获得了Naïve-rbESC，其生物学特性与mESC Naïve态相似，其 Naïve态的维持高度依赖bFGF、Erk、GSK3和PKC信号的协同作用。研究显示抑制PKC信号捕获的Naïve态可能与2iL-Naïve特征不同。该研究为建立真正具有种质传递的rbESC系奠定了基础，将有助于家兔作为模式动物在生物学及医学领域的应用。