家兔胚胎干细胞与其种质传递机制的研究

The purpose of this study is to establish germline competent rbES cell lines. Rabbit is considered as a good animal model for study of many human diseases. It has not been widely used, however, due to the fact that there are no germline competent rabbit embryonic stem (rbES) cell lines. Rabbit ES-like cell lines were established almost 20 years ago. Following injection into blastocysts, these cells formed coat color chimeras, but did not transmit into the germ line. There is not a research team to date in the world that can prove the totipotency of rbES, that is, rbES can continue to differentiate into the specific germ cells, and finally have the potentials to transmit into the germplasm (germline). In this study, we will study the mechanisms controlling rbES self-renewal and further their ability of germline transmission. In our preliminary study, we have (1) established rabbit embryonic fibroblast (REF) feeder cells, and synthesized recombinant rabbit leukemia inhibitor factor (rbLIF); (2) examined the effects of the origin of REF feeder cells and LIF on the derivation and maintenance of rbES cells; (3) identified several important factors/signal pathways governing rbES cell self-renewal; and (4) established putative rbES cell lines. We believe that our project will be supported by the National Natural Science Fund based on the scientific merits of research. We propose to study on the following researches: (1) to explore the molecular mechanism for rbES derivation and maintenance, and the signal pathways that control characteristics of authentic rabbit embryonic stem cells; (2) to develop specific medium system suitable for a unique rbES derivation, and establishing more rbES lines, including transgenic GFP fluorescent rbES; (3) to analyze of the cellular and molecular characteristics in vitro of those existing rbES, and any new rbES lines to be derived; Finally, we expect (4) to establish at least one line of rbES that possesses the potential of germline transmission in vivo, and to analyze of its mechanisms to maintain stem cell totipotency.

本研究旨在建立具有种质潜能的家兔胚胎干细胞（rbES），研究其自我更新与种质传递的机理。家兔是研究许多人类疾病的优势动物模型。rbES 建立已近20年。但至今，全世界仍无一团队证明rbES能继续分化成特殊生殖细胞, 具备种质传递的潜能，使家兔模型未能广泛应用。目前对rbES的自我更新和维持的机制还存在争议。我们的前期研究证实：（1）兔胚饲养层细胞(REF)与重组家兔白血病抑制因子(rbLIF）对rbES的获取与维持具有促进作用；（2）确定了维持rbES自我更新的几个重要信号通路分子。我们推测控制rbES自我更新的信号通路将极大地影响干细胞的种质传递能力。我们将创新性地利用REF、rbLIF和特定的rb-2iF培养基获得新的rbES，加深对rbES自我更新和细胞分化调控通路的认识；利用GFP标记的rbES，研究其体外与体内分化的态势，并揭示rbES在嵌合体中传递成生殖系细胞的规律。

家兔是研究许多人类疾病的优势动物模型。家兔胚胎干细胞（rbESC） 建立已近20年。但至今，全世界仍无一团队证明rbESC能继续分化成生殖细胞, 具备种质传递的潜能，限制了家兔模型广泛应用。我们检测了rbESC自我更新相关的信号通路中的多种因子，结果显示bFGF通过激活FGF信号通路对rbESC细胞的自我更新中起着关键性的作用，Noggin通过抑制BMP信号通路从而抑制rbESC分化。我们利用不同浓度的细胞因子研究出rbESC培养基iFLY，确定了维持rbECS自我更新的最佳剂量，LIF、bFGF、Noggin的浓度分别在103IU/ml、40ng/ml、400 ng/ml时培养效果最佳。利用玻璃化冷冻囊胚获取了rbESC细胞系，与从新鲜的囊胚中获取rbESC的效率相比无显著差异，GFP标记的rbESC细胞系可产生嵌合体兔仔。表观修饰与多能性关键因子的表达对植入前胚胎获得发育多能性至关重要。我们系统地研究了植入前兔胚胎在体内发育过程中组蛋白H3K4me3、H3K27me3修饰水平及Nanog表达动态模式，为分离rbESC的分离提供数据支撑。在此基础上探索了Naïve态rbESC的分离，通过小分子调制相关信号通路，分离到Naïve-like态rbESC。实验结果显示在3i条件下，抑制GSK3 (CHIR99021)、MEK (PD0325901)、PKC (Gӧ6983)信号通路，分离到“Dome-shaped” 形态rbESC，证明“Dome-shaped”形态维持与抑制PKC信号直接相关。目前rbESC领域相关研究进展较少，我们的研究结果填补了rbESC研究的空白，确立了naive态rbESC维持所需的信号通路条件、冷冻胚胎是否能分离到rbESC、并用GFP标记了rbESC，为干细胞胚胎嵌合验证提供了遗传标记材料等。同时我们证实在家兔中可以建立起Naïve态rbESC，为后续研究提供了珍贵的研究数据和材料，为后续突破奠定了基础。