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 a good animal model for the study of many human diseases. It has not been widely used, however, due to the fact that there are no germline competent embryonic stem (ES) cell lines in this species. Rabbit ES-like cell lines were established almost 20 years ago. Following injection into blastocysts, these cells formed coat color chimeras, but did not colonize the germ line. However, there is no research team, to date, in the world that can prove the totipotency of rabbit ES (rbES), that is, rbES can continue to differentiate into the specific germ cells, and finally have the potentials to transmit into the germplasm (germline). 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/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 pathway that controls 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）确定了控制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。本研究将创新性地利用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，研究其在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。

本项目们检测rbES自我更新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rbES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rbES分化，CHIR99021通过抑制GSK3信号通路而激活Wnt通路，对rbES起到促进生长和抑制分化的作用。我们利用不同浓度的细胞因子调配成rbES培养基，确定了维持rbES自我更新的最佳剂量，bFGF、Noggin、CHIR99021的浓度分别在40ng/ml、400 ng/ml、6 μM时培养效果最佳。根据这些因子的最佳剂量，我们调配出适合家兔干细胞培养的特定rb-2iF培养基。