纺锤体组装检查点介导胚胎染色体嵌合自我校正机制研究

Mosaic chromosomal composition is common during preimplantation development of human embryos. A part of mosaic embryos are of the capacity to develop euploidy embryos and give live births, indicating potential self-correction mechanism in embryogenesis. Our previous study on peri-blastocyst cells which were not compacted with the main cell mass of the embryo at morula stage suggested clonal depletion is a significant mechanism for embryo correction of mosaicism. The spindle assembly checkpoint (SAC) plays a vital role in chromosomal segregation and cell division. Recent research revealed that mitotic spindle disruption can activate SAC and induce apoptosis (clonal depletion). However, the mechanism and molecular basis are unclear. The current study is aiming at discover the molecular mechanism of embryo self-correction from the aspect of clinical retrospective analysis, cell line model, and animal model: retrospective study on clinical pregnancy outcomes and chromosomal composition of fetal cells or tissue derived from mosaic embryos to validate the association between mosaicism and reproduction outcomes, as well as elucidate the frequency and spectrum of mosaic chromosomes in human embryos; research through artificial mosaic cell lines to unravel the basis in regulating proliferation and apoptosis of aneuploidy cells and dynamics of mosaic ratio by the SAC pathway; and establishment of mouse mosaic embryo model and clonal embryo model derived from single blastomeres to discover the molecular mechanism and cellular basis that the SAC pathway induce embryo self-correction by postponing the metaphase-anaphase transition of aneuploidy cells and inducing their arrest/apoptosis. This study is expected to renew the current understanding of mosaic embryos and their potential, and further, to provide theoretical basis and new insight into human embryo development and embryo assessment and selection in assisted reproduction.

染色体嵌合是人类胚胎发育过程中的普遍现象。部分嵌合体胚胎可发育为整倍体并活产，提示发育过程中存在嵌合校正机制。前期对嚢胚腔外细胞的研究提示克隆耗竭可能是嵌合校正的重要机制。纺锤体组装检查点（SAC）是控制染色体分离和细胞分裂的重要保障。最新研究证实纺锤体扰乱可激活SAC介导异常细胞凋亡（克隆耗竭），但其分子机制不明。本项目拟从临床回顾性分析、细胞模型、动物胚胎模型三个层面开展研究：通过临床回顾性分析明确人类胚胎嵌合体的频谱及嵌合体胚胎的发育潜能和妊娠结局；通过嵌合细胞模型，阐明SAC调控非整倍体细胞增殖与凋亡及染色体嵌合比例动态变化的分子基础；构建嵌合小鼠胚胎模型及单细胞克隆胚胎模型，阐明SAC通过延迟非整倍体细胞分裂中期-后期转换并诱导其阻滞/凋亡以校正胚胎嵌合的分子机制和细胞学基础。本研究有望革新对嵌合体胚胎发育的现有认识，为理解人类个体发育及辅助生殖胚胎潜能评估提供理论依据和新思路。

染色体嵌合是人类胚胎发育过程中的普遍现象。部分嵌合体胚胎可发育为整倍体并活产，提示发育过程中存在嵌合校正机制。我们前期对未能完成致密化的嚢胚腔外细胞研究提示克隆耗竭可能是嵌合校正的重要机制。纺锤体组装检查点（SAC）是控制染色体分离和细胞分裂的重要保障。最新研究证实纺锤体扰乱可激活SAC介导异常细胞凋亡（克隆耗竭），但其分子机制不明。本项目从临床回顾性分析、细胞模型、动物胚胎模型三个层面开展研究。通过临床回顾性分析，明确人类胚胎嵌合体的频谱及嵌合体胚胎的发育潜能和妊娠结局，胚胎嵌合体可累及全染色体组中的任何染色体，各染色体间未见显著差异，与整倍体胚胎相比，嵌合体胚胎的植入率、临床妊娠率、持续妊娠/活产率显著降低，流产率显著升高。通过染色体嵌合组织和嵌合细胞模型研究，探明SAC调控非整倍体细胞增殖与凋亡及染色体嵌合比例动态变化的关键因子及其表达差异与互作网络模式。SAC通路中TPR、USP44、DYNC1LI1、MAD2L1BP等基因在不同代次和不同染色体嵌合比例细胞系中差异表达提示其可能参与调控非整倍体细胞增殖与凋亡，且SAC通路可能与细胞自噬、细胞周期、细胞骨架和线粒体动态等相关生物过程协同作用。构建染色体嵌合小鼠胚胎模型，通过胚胎单细胞基因组与转录组并行测序及胚胎免疫荧光分析，从单细胞水平上验证SAC通路中Tpr、Dync1li1、Mad2l1bp等基因在整倍体与非整倍体细胞间的差异表达模式和调控网络，提示SAC可能通过延迟非整倍体细胞分裂中期-后期转换并诱导其阻滞/凋亡以校正胚胎染色体嵌合。本项目初步阐明了胚胎早期发育过程中染色体非整倍体嵌合的潜在校正机制，为深入研究早期胚胎核质动态精细调控机制及多组学互作网络奠定基础，有助于革新我们对染色体嵌合体胚胎发育的现有认识，深入理解人类生殖和个体发育过程，为全面评估胚胎发育潜能以改善辅助生殖临床妊娠结局提供理论依据和新思路。