PLAC8在人类早期胚胎发育与植入过程中的作用及机制研究

Implantation failure is the primary cause for in vitro fertilization (IVF) failure. Placenta-specific 8 (PLAC8) was shown to be a key molecule in early mammalian embryonic development and in the process of embryonic implantation, however, little is known about PLAC8 expression in human embryos and embryonic implantation. The applicant preliminary work suggests the presence of PLAC8 protein in the cytoplasm of all human oocytes and embryos preceding implantation, and after the blastocysts implanting and invading endometrial stromal cells, PLAC8 protein was located in the nucleolus, relating to the state of embryonic implantation. Using embryo-endometrial stromal cell co-culture models, this project is planed to clear the effect of PLAC8 on human early embryo development and blastocyst implantation process in vitro, and to explore its possible molecular mechanisms by interference and overexpression PLAC8 molecules of human abnormal fertilized embryo. Furthermore, we plan to use human blastocyst trophoblastic single-cell transcriptome sequencing and difference transcriptome analysis to observe the correlation of PLAC8 expression and pregnancy rate, to preliminary find the upstream and downstream molecules of PLAC8, and to explore PLAC8 role in the human early embryo development and implantation process. The knowledge of our study will provide important experimental basis on the development and implantation mechanism of human embryo, and will be benefit for looking for a new biological marker indicating potential of embryonic implantation.

植入失败是限制人类体外受精胚胎种植率的关键问题。已知胎盘特异性抗原8（PLAC8）是参与调控哺乳动物胚胎发育和植入的重要分子，但其在人类胚胎植入中的作用尚不清楚。申请人前期工作发现PLAC8在人类早期胚胎中表达，且在人类囊胚体外植入过程中的表达及定位变化与胚胎植入状态相关。本项目拟在前期工作基础上，利用所建立的人胚胎-子宫内膜基质细胞共培养模型，通过在临床废弃人早期胚胎上干扰和过表达PLAC8，明确PLAC8对人类早期胚胎发育及囊胚体外植入过程的影响；进一步采用单细胞转录组测序技术检测拟进行植入前遗传学筛查的人类囊胚滋养层细胞，分析PLAC8表达与胚胎体内移植后妊娠率的相关性；同时比较胚胎移植后妊娠成功与否的PLAC8相关转录组差异，寻找PLAC8上下游分子，初步探讨PLAC8参与调控人类胚胎种植的可能机制。项目将为阐明人类早期胚胎着床种植机理、寻找有效标志胚胎着床潜能的关键分子提供依据。

植入失败是限制人类体外受精胚胎种植率的关键问题。已知胎盘特异性抗原8（PLAC8）是参与调控哺乳动物胚胎发育和植入的重要分子，但其在人类胚胎植入中的作用尚不清楚。前期工作发现PLAC8在人类囊胚体外植入过程中的表达及定位变化与胚胎植入状态相关，预示着其潜在的生物学功能，然而其具体作用和调控机制尚不清楚。.研究一 成功构建了 PLAC8 的慢病毒敲降体系； 慢病毒可以成功转染鼠胚和人类早期胚胎，使用1×107TU/ml诱导PLAC8 RNAi的慢病毒载体感染人第2-3天的3PN胚胎，敲降组胚胎卵裂率（6.5）较阴性对照组（空载体组）显著降低（10.57±1.87）；敲降组胚胎停止发育率（86%）显著高于阴性对照组（53%）。慢病毒干扰载体成功转染人胚胎干细胞hESCs，阴性对照组与敲降组的 PLAC8 mRNA的相对表达量分别是1.00±0.06，0.41±0.06，敲降组 PLAC8 mRNA表达量低于阴性对照组（P<0.05）, 具有统计学意义；Western blot实验结果显示：敲降组表达下调，进一步证明慢病毒介导干扰质粒能敲降PLAC8蛋白表达。敲降组与阴性对照组相比细胞增殖相关的基因CCND1和Ki67均下调，敲降组的CCND1 mRNA和ki67 mRNA的相对表达量分别是0.46±0.088和0.41±0.174，具有统计学意义；同时敲降组较阴性对照组 cyclinD1、PCNA蛋白下调，阴性对照组和敲降组的cyclin D1蛋白表达量分别1.32±0.18和0.78±0.11，PCNA蛋白表达量分别是1.69±0.11和1.41±0.08，经分析差异具有统计学意义。流式细胞术检测敲降组凋亡率比阴性对照组增高。.研究二 成功收集经过体外受精后的小鼠囊胚；经过实验发现运用慢病毒的滴度在1×107TU/ml转染小鼠囊胚最合适；成功的把转染后的囊胚移植到小鼠的子宫内，2.5天后发现PLAC8 敲降组囊胚附植率低于阴性对照组。.通过本研究首次成功构建PLAC8 RNAi的慢病毒载体，证实了PLAC8影响人早期胚胎的发育；细胞实验进一步验证敲降人胚胎干细胞PLAC8基因的表达，会降低细胞增殖表达并促进细胞凋亡；敲降小鼠囊胚的PLAC8基因表达会导致胚胎的附植率降低。该研究结果可为为进一步阐明人类早期胚胎的生长发育和着床种植机理提供重要的实验依据。