O-GlcNAc糖基化修饰通过代谢重编程抑制ART小鼠胎盘血管生成的机制研究

Assisted reproductive technology (ART) results in abnormal placental angiogenesis in humans and many animals, but its mechanism is unclear. We previously found that the level of angiogenesis in placental labyrinth layer was significantly decreased, and the levels of O-GlcNAc transferase (OGT) and HIF-1α protein were also significantly decreased by ART in mouse placenta. Considering that the OGT could regulate the glycometabolism by modifying the pyruvate kinase M2 (PKM2) with O-GlcNAc which affects HIF-1α stability, we proposed the hypothesis that down-regulation of ART placental OGT expression could cause the decrease of O-GlcNAc modification level of PKM2, which increased the enzyme activity and the level of Krebs cycle, and finally promoted HIF-1α degradation and inhibited placental angiogenesis. On the basis of the previous work, firstly ART mouse model and mouse cell line will be used to reveal the mechanism of inhibiting placental angiogenesis by down-regulation of OGT that induced by ART. Secondly, the molecular mechanism of the down regulation of OGT expression in ART placenta will be elucidated by ChIP technique. Finally, the intervention strategy of preventing and treating placental angiogenesis abnormality of ART will be explored by using small molecule Thiamet G. The completion of the project will reveal the new mechanism of abnormal placental angiogenesis induced by ART and provide scientific basis and experimental support for the abatement of adverse pregnancy outcomes originating from ART.

ART致人类及多种动物胎盘血管生成异常，但其发生机制不明。我们前期发现，ART致小鼠胎盘迷路层血管生成水平显著下降，且O-GlcNAc糖基转移酶（OGT）与HIF-1α蛋白水平显著下调。鉴于OGT可通过O-GlcNAc修饰丙酮酸激酶PKM2调控糖代谢影响HIF-1α稳定性，我们提出ART胎盘OGT表达下调，致PKM2的O-GlcNAc修饰水平降低、酶活性升高，三羧酸循环代谢增强，最终促使HIF-1α降解，抑制胎盘血管生成的假说。本课题拟在前期工作的基础上，首先，利用ART小鼠模型和细胞系，揭示ART致OGT下调，抑制胎盘血管生成的发生机制；其次，通过ChIP技术，阐明ART致胎盘OGT表达下调的分子机制；最后，利用小分子Thiamet G，探索防治ART胎盘血管生成异常的干预策略。本项目的完成，将揭示ART致胎盘血管生成异常的新机制，为消减ART源不良妊娠结局提供理论和实验支持。

在辅助生殖临床中，新鲜胚胎移植周期不良妊娠结局的发生风险显著升高，胎盘异常是导致不良妊娠结局的重要原因。本项目，我们利用小鼠胚胎移植模型排除辅助生殖技术（assisted reproductive technology, ART）中超促排卵对卵子及早期胚胎的影响，研究ART超促排卵通过影响子宫内膜对胎鼠发育及胎盘功能的影响。首先，我们发现，ART超促排卵致小鼠胚胎植入率、胎鼠体重及胎盘血管密度显著降低；其次，发现ART超促排卵致小鼠蜕膜重量显著下降，蜕膜组织中促血管生成因子的表达水平均显著下调；再次，发现ART超促排卵致蜕膜组织糖酵解关键基因HIF-1a及PKM2的表达水平显著下调；进而研究发现，ART超促排卵致蜕膜组织O-GlcNAc转移酶（O-GlcNAc transferase, OGT）的表达水平、蛋白O-GlcNAc修饰水平显著下调。我们利用人类子宫内膜基质细胞，进一步揭示了ART超促排卵致蜕膜组织血管生成因子水平下调的发生机制。我们首先发现，蜕膜化过程中，OGT的表达水平、蛋白O-GlcNAc修饰水平显著升高，糖酵解关键基因HIF-1α等的表达水平均显著上调。进而发现，利用小分子抑制剂抑制OGT活性，可抑制子宫内膜基质细胞HIF-1α及血管生成相关因子的表达。我们的研究表明，ART超促排卵可通过抑制OGT的表达，进而影响糖酵解关键基因HIF-1α的表达，致血管生成相关因子的表达水平下调，最终导致ART胎盘血管的密度下降。本项目，通过胚胎移植动物模型，排除了超促排卵对卵母细胞及早期胚胎发育的影响，发现ART超促排卵可通过影响子宫内膜OGT的表达，进而影响HIF-1α的表达及稳定性，导致血管生成相关因子的表达水平下调。这可能是新鲜移植周期，ART胎盘血管生成水平下调的重要原因。本项目的完成，为辅助生殖临床超促排卵所导致的胎盘生长和发育异常提供了新的理论依据。