Dlk1在猪黄体化颗粒细胞中的作用与分子机制研究

Dlk1, a noncanonical ligand of Notch pathway, can regulate the proliferation and differentiation of numerous types of cells. In our previous work, Dlk1 A was found to be highly expressed in luteinized porcine granulosa cells (LGCs), its expression was stimulated and inhibited by hCG and activin, respectively. In addition, Dlk1 was reported to be located in the large luteal cells, also known as the granulosa-lutein cells. To further demonstrate the regulatory roles and moleuclar mechanisms of Dlk1 in development and function of the granulosa-lutein cells, this study plans to investigate the spatio-temporal expression pattern of Dlk1 and Notch proteins in porcine corpora lutea. In addition, in vitro cultured LGCs will be used as model of Dlk1 regulation of cell proliferation, apoptosis and synthesis of progesterone. Rescue and inhibiton of the candidate dlk1 pathways by NICD over-expression, treatment of pathway inhibitors DAPT, U0126 and LY294002 will be performed. Results of the above studies will help to expand our knowledge on CL development, and will help to identify new molecular target to control CL function and development, so to extend new dimention of regulation of animal reproductive performance.

Dlk1是Notch通路的非典型配体，参与调控许多细胞的增殖和分化。在卵巢中，Dlk1蛋白定位于卵泡膜细胞和大黄体细胞。申请者前期研究表明，猪Dlk1有A、B、C和C2四种剪切体，其中Dlk1 A在猪黄体化颗粒细胞（LGCs）中高表达，并且hCG和Activin分别促进和抑制Dlk1 A的表达。为进一步探索Dlk1在猪黄体中的作用，首先研究Dlk1与Notch通路蛋白在黄体发育不同时间和不同细胞的时空表达模式。然后以猪LGCs为细胞模型，研究Dlk1过表达和干扰对大黄体细胞的增殖、凋亡、P4合成和促血管因子表达等的影响。最后通过过表达NICD、加入通路抑制剂DAPT、U0126和LY294002来挽救或抑制Dlk1候选通路，从正反两方面验证Dlk1调控LGCs功能的分子机制。通过以上研究，可以丰富猪黄体发育和功能调控的理论，为调控黄体功能提供新的靶点，从而扩展动物繁殖调控技术新领域。

Notch信号通路参与调控哺乳动物卵泡发育和黄体功能，但具体的功能在各报道之间存在矛盾之处，并且相应的分子机制有待于深入研究。项目申请人前期研究发现Notch信号通路的非典型配体Dlk1表达于黄体化颗粒细胞（LGCs），并且在黄体化过程Dlk1 A表达显著上升，但Dlk1是否发挥调控LGCs功能及其机制未知。因此，为进一步探索Notch信号通路以及其配体Dlk1在LGC的功能，项目申请人首先在LGCs过表达Dlk1两种剪切体Dlk1 A和C2，发现Dlk1过表达并不显著影响LGCs的细胞增殖和类固醇激素P4和E2的表达。接着，利用Notch通路抑制剂DAPT处理LGCs，发现DAPT可以剂量和时间依赖的促进P4的分泌，定量分析发现DAPT通过上调NR5A2和NR2F2的表达促进StAR和NPC1的表达。干扰NR5A2和NR2F2的表达，可以抑制DAPT处理上调的StAR和NPC1表达，下调P4分泌。申请人还发现，干扰NR5A2和NR2F2处理上调Notch3的表达。这表明Notch通路和转录因子NR5A2和NR2F2可以相互抑制，NR5A2和NR2F2发挥促进StAR和NPC1表达和P4分泌的作用。进一步的，干扰NR5A2和NR2F2后转录组分析发现，NR5A2和NR2F2在LGCs可能发挥调控氧化应激、羟基胆固醇和胆汁酸代谢合成的作用。以上发现极大扩展了Notch信号通路调控猪颗LGCs的功能以及相应分子机制的认识，丰富了哺乳动物卵泡发育的理论。同时，有望利用Notch信号通路调控LGCs类固醇激素合成的特性，开发以Notch信号通路为靶点的哺乳动物类固醇激素调控新药物或者动物繁殖调控新技术。