Tet蛋白在植入前胚胎DNA去甲基化过程中的作用及其影响因素的研究

About 40% of the preimplantation embryos in ART were failed to develop in the process of in vitro culture. Abnormal demethylation may be one cause, which was found to be a very important process during the formation of normal DNA methylation patterns, but the mechanism is unclear. Previous study showed that Vitamin C (VitC) can promote the demethylation process during the reprogram of stem cells. Tet protein family, a new family of enzymes, can modify 5mC through oxidation to 5-hydroxymethylcytosine (5hmC) in active demethylation pathways. Our preliminary data showed that expression of Tet proteins and 5hmC level was significantly down-regulated in the blastocyst compared with the viable blastocyst. Whether Tet protein and demethylation are key factors in human embryo development? Can adding VitC improve the embryo quality through promoting demethylation effect? In the present study, human preimplantation embryos were collected and the global DNA demethylation and the expression of Tet proteins will be detected in pre-implantation embryos at different developmental stages. The relationship between down-regulation of Tet proteins family and abnormal embryonic development will be explored. Then VitC will be added to the embryonic in vitro culture system, and we aim to explore whether VitC play a role in the regulation of demethylation mediated by Tet proteins family, ultimately promote the early embryo development. We hope that this study can provide more scientific basis on the role of Tet protein family in DNA methylation patterns of preimplantation embryos and the optimization of in vitro culture system.

辅助生殖技术中约40%的胚胎在体外培养过程中停止发育，DNA去甲基化受阻是导致胚胎停育的重要原因，但具体机制未明。Tet蛋白可能通过催化5-甲基胞嘧啶（5mC）羟化为5-羟甲基胞嘧啶（5hmC）介导去甲基化，而维生素C（Vit C）可加速去甲基化作用。我们前期研究显示在停育的人囊胚中Tet蛋白表达及5hmC含量均下降。Tet蛋白调控去甲基化是否是影响人胚胎发育的关键因素？若添加Vit C是否可加强去甲基化作用从而提高胚胎质量？本项目拟通过单细胞RNA-seq技术，免疫荧光，焦磷酸测序等技术检测植入前胚胎不同阶段Tet蛋白表达变化及5hmC的水平，构建Tet基因敲除鼠胚模型，探索Tet蛋白在早期胚胎发育和DNA去甲基化中的作用及分子机制，分析Vit C与去甲基化和Tet蛋白的相关性，为优化人胚胎培养体系提高胚胎发育潜能提供理论依据。

为了探索早期胚胎停止发育和DNA甲基化之间的关系，本研究检测了人植入前胚胎整体DNA甲基化水平及Tet蛋白表达的变化，并建立了Tet基因敲除的动物模型来进一步验证DNA甲基化水平与Tet蛋白变化之间的因果关系，尝试在胚胎培养体系中添加维生素C是否可调控Tet蛋白介导的去甲基化作用从而促进早期胚胎发育，具体如下：1）随着胚胎的生长发育，由原核期至囊胚，人植入前胚胎自原核期至囊胚期5mc水平逐渐下降，5hmc水平不断升高，即DNA甲基化水平逐渐下降，囊胚期达最低点；2）在体外停止发育的人早期胚胎中，其Tet1和Tet2蛋白水平下降、DNA甲基化水平异常增高，其可能机制是Tet蛋白表达不足以致胚胎的去甲基化过程受到影响，从而影响胚胎发育；3）建立Tet基因敲除的小鼠（DKO）模型，收集野生型（WT）小鼠和DKO小鼠在体外各发育阶段的鼠胚，检测发现DKO小鼠胚胎不能表达Tet1/2蛋白，DNA甲基化水平更高，D3胚胎比例更低，胚胎无法发育至囊胚；4）当在野生型小鼠胚胎体外培养体系中添加VitC后，胚胎停育的比例下降，经过VitC处理的胚胎其Tet蛋白表达更高，DNA甲基化水平更低。同时，也延伸出更多的科学问题有待解决：本研究体外培养液中添加VitC，其浓度的设定是来源于参考干细胞研究，而胚胎对培养环境的变化十分敏感，需进一步研究探索合适的VitC浓度，来提供可能应用于临床的安全性和有效性数据。因此，希望通过优化胚胎培养体系达到优化胚胎结局的目的，甚至为优生优育提供可能的科学依据。