雌激素与小鼠植入前胚胎发育过程中表观遗传模式正确建立的相关性研究

Previous studies suggested that estrogen and epigenetic modification play important roles in mouse preimplantation embryo development and implantation, but the correlation between estrogen and the establishment of epigenetic modification pattern in this process remains unclear, especially the function of estrogen. Based on our previous studies, we propose that estrogen control the epigenetic modification, such as DNA methylation and histone modification, through modulating the transcription regulation of epigenetic modifying enzyme genes to participate in mouse preimplantation embryo development. Using culture system with estrogen, promoter assay system, real-time PCR, immunofluorescence microscopy, Western blot and enzyme activity analysis, we will examine the effect of estrogen on the mRNA expression, protein, and promoter activities of epigenetic modifying enzyme genes and enzyme activity in mouse IVF and cloned embryos. Electrophoretic Mobility Shift Assay (EMSA), Chromatin Immunoprecipitation(ChIP) and RNAi technology will be further performed to illustrate the molecular mechanisms of transcription regulation of epigenetic modifying enzymes regulation by estrogen and its receptors. Furthermore, ER gene knockout cells-cloned embryo was constructed to further in vivo validate the effect of estrogen on the cloned embryo development and epigenetic modification. The study aims to clarify the correlation between estrogen and the establishment of flawless epigenetic pattern during mouse preimplantation embryo development, and the results will provide new insight into the function of estrogen and epigenetic modification on the embryo development, and basis for promoting the rate of cloned embryo development and assisted reproduction.

雌激素和表观遗传修饰对小鼠植入前胚胎发育有重要影响，但鲜有揭示二者在胚胎发育过程中的相关性特别是雌激素功能的报道。结合前期研究，我们提出雌激素可能通过影响植入前胚胎表观遗传模式的建立和发育相关因子的表达，参与胚胎发育的观点，并拟以小鼠体外胚和体细胞克隆胚等植入前胚胎为材料，从以下3方面论证我们的设想：1）在培养基中添加雌激素，利用qPCR、免疫荧光、Western杂交、酶活力分析等方法，研究雌激素对表观遗传修饰酶、重要发育基因表达和酶活力的影响；2）通过启动子分析、ChIP和EMSA等方法揭示雌激素对表观遗传修饰相关酶基因转录调控的分子机制；3）通过RNA干扰抑制ER基因表达和以ER基因敲除的稳转系细胞为供体构建克隆胚，在体验证雌激素对表观遗传模式正确建立的影响和相关性。本研究将阐明雌激素在小鼠植入前胚胎发育过程中功能和作用机制，为提高克隆胚发育率和实验动物进一步模式化奠定基础。

雌激素对小鼠植入前胚胎发育有影响，雌激素缺失引起后期胚胎植入率的低下，但迄今为止并未从分子机制上揭示产生这一现象的原因。DNA甲基化和组蛋白修饰等表观遗传修饰在小鼠胚胎发育过程中有重要作用。二者在小鼠胚胎发育过程中通过怎样的分子机制进行偶联未见报道。本项目从分子机制和和表观遗传修饰模式建立的角度入手，探讨了二者偶联及其作用的分子机制，揭示了雌激素通过与受体形成复合物物理性结合在表观遗传修饰酶基因启动子上，调节此类基因表达水平，影响表观遗传修饰酶活力进而参与小鼠植入前胚胎发育过程中表观遗传修饰模式的正确建立，加深对雌激素在哺乳动物植入前胚胎发育过程中作用的认识，为后期提高克隆胚的发育率、胚胎植入和克隆动物的成功率奠定基础，在人工辅助生殖技术（体外培养环节）具有重要的应用前景。此外我们还对植物性雌激素、环境类雌激素影响小鼠卵巢发育的分子机制进行了探讨，为环境保护和人类健康提供了基础的理论依据；通过单细胞转录组分析，我们还构建了不同供体来源的小鼠植入前克隆胚胎mRNA和lncRNA精细图谱，发现表观重编程不彻底是导致基因再激活和重编程效率低的主要原因，为核重编程技术在再生医学和农业等行业的推广和应用奠定基础。