ZEB1通过促进上皮-间质转化影响子宫内膜容受性在胚胎着床中的机制

The failure of embryo implantation is the main cause of recurrent spontaneous abortion and infertility，and is closely associated with endometrial receptivity. The epithelial-mesenchymal transition (EMT) of endometrium epithelial cell (EEC) is crucial for the remodeling of EEC. Zinc finger transcription factor ZEB1 could promote EMT of tumor cells and mediate the invasion and metastasis of tumor. However, whether ZEB1 could promote EMT of EEC and alter endometrial receptivity is still unclear. Our preliminary results showed that, after knockdown of ZEB1 with shRNA in EEC in vitro, the proliferation ability was decreased, while the expression of E-cadherin was up-regulated and that of vimentin was down-regulated (both of which are key EMT genes), suggesting that ZEB1 could alter EEC receptivity through modulation of EMT, and play a role in the initial stage of embryo implantation. By using in vitro cell culture model, in vitro embryo implantation model and animal model, the present project plans to study the followings: 1) the effects of ZEB1 on cell function and EMT in EEC in vitro; 2) the effects of ZEB1 on embryo implantation in an in vitro model; and 3) the expression pattern of ZEB1 in endometrium in pregnant mice, and its effect on mouse embryo implantation. Studying the mechanisms by with ZEB1 alters endometrial receptivity through promoting EMT of EEC in embryo implantation, is hopeful to provide new knowledge for the mechanisms of pregnancy failures.

胚胎着床失败是复发性流产和不孕症的主要原因，与子宫内膜容受性密切相关。子宫内膜上皮细胞（EEC）的上皮-间质转化（EMT）对EEC重塑至关重要。锌指转录因子ZEB1可促进肿瘤细胞EMT，介导肿瘤侵袭和转移，但其能否促进EEC的EMT改变子宫内膜容受性尚不明确。我们前期结果显示：离体EEC中shRNA下调ZEB1表达后，细胞增殖能力降低，EMT关键基因E-钙粘蛋白表达上调、波形蛋白表达下调，提示ZEB1可通过调节EMT，改变EEC容受性，参与胚胎着床初始过程。本项目拟借助离体细胞培养模型和胚胎着床模型及动物模型，研究：1)离体EEC中ZEB1对细胞功能和EMT的影响；2)离体胚胎着床模型中ZEB1对胚胎着床的影响；3)ZEB1在妊娠小鼠子宫内膜的表达规律和对小鼠胚胎植入功能的作用。研究ZEB1通过促进EEC的EMT改变子宫内膜容受性在胚胎着床中的机制，有望为妊娠失败的机理提供新的理论知识。

哺乳动物胚胎着床过程，子宫内膜均对胚胎具有容受性，子宫内膜容受性关系着胚胎着床的成功与否。上皮间质转化（EMT）在很多生物学过程中起很重要的作用。已证实在雌性生殖系统中，EMT过程也广泛存在。锌指转录因子ZEB1能与E-钙粘蛋白基因的E-box区结合，从而抑制E-钙粘蛋白的转录，诱导细胞发生EMT，增强细胞的迁移和侵袭能力。ZEB1在EMT中起着重要作用。有研究证实子宫内膜和子宫肌层上也有ZEB1的表达，但胚胎着床和发育过程中的EMT是否与ZEB1有关，其作用和机制尚不明确。.本研究利用RNA干扰技术构建ZEB1的shRNA慢病毒载体，同时建立人体外胚胎植入体外模型和妊娠小鼠模型，对ZEB1在胚胎植入过程中的作用进行初步研究。研究结果发现ZEB1在分泌期的人子宫内膜及小鼠着床的窗口期的子宫内膜均高表达，伴随着E-cadherin和Vimentin表达改变，提示在胚胎着床过程中发生了EMT。敲低ZEB1表达明显抑制了体外容受性子宫内膜细胞RL95-2细胞的增殖、迁移和DNA复制能力。此外，RL95-2细胞中敲低ZEB1敲低后，显著增高了E-cadherin表达，降低了Vimentin表达，并使模型胚胎JAR球体对RL95-2细胞的粘附率显著下降。上述结果提示ZEB1在子宫内膜中，可通过促进EMT过程改变子宫内膜容受性，促进胚胎植入；该作用可能是通过下调E-cadherin和上调Vimentin。我们还发现宫角注射ZEB1-shRNA，可明显降低小鼠胚胎的着床率，该结果在在体水平证明了ZEB1对胚胎着床具有重要调节作用。.综上所述，本研究在细胞、组织、整体动物水平上证实了ZEB1在胚胎着床中的功能和分子机制，对胚胎着床的生理病理过程提供了新的理论依据。同时ZEB1反义寡核苷酸可以特异性地下调ZEB1的表达，从而起到抑制体外胚胎着床的作用，这为人工避孕以及提高体外人工受精的成功率提供了新的思路