Galectin-1调控滋养层干细胞的增值和分化及其在母-胎对话中作用机制的研究

Trophoblastic proliferation and differentiation in an orderly manner play a critical role in the embryonic implantation, placentation and maintencance of early pregnancy. The embryo implantation failure and early pregnancy loss are intensively related with poorly trophoblastic proliferation and differentiation. Our previous proteomics study on human EPL chorionic tissues found that galectin-1 was downregulated significantly in trophoblastic cells of EPL when compared with the controls, which suggestting that galectine-1 has an important role in early placental development and maintenance of pregnancy. In this protocol, we aim to reveal the characteristics of spatial-temporal expression and epigenetic regulative pattern of galectin-1 and related molecules during the development of trophoblastic cells, placentation and early pregnancy by using mouse trophoblast stem cells as a proliferation-differentiation model , combination with the in vitro gene control techniques, embryonic micromanipulation, pseudo-pregnancy mouse model and stress-induced mouse models. Ultimately, we aim to determinate the pathologic mechanism of EPL, and provide valuable information and seek possible direction for cellular and molecular treatment.ferentiation model stimulation the development process of trophoblast cell, combination with the in vitro gene expression and regulation, embryo micromanipulation and related animal models, complete reveal of galectin-1 characteristics of spatial-temporal expression, expression regulation patterns, and related important molecular mode of action in trophoblastic proliferation/differentiation. With a mouse model, investigate the role of galectine-1 in embryo implantation and early pregnancy maintenance. Ultimately reveals the mechanisms of embryo implantation and EPL, provide valuable basis for seeking treatment methods.

有序的滋养细胞增殖、分化进程在胚胎植入、胎盘形成及妊娠维持中起着重要的作用。胚胎着床失败及早期妊娠丢失等与滋养细胞增殖和分化不良密切相关。我们前期蛋白质组学研究发现，半乳糖凝集素-1（Galectin-1）在人EPL滋养细胞中表达较正常对照显著降低，提示Galectin-1在早期胎盘发育、母-胎对话中扮演了重要角色。本项目拟应用小鼠滋养层干细胞增殖-分化模型模拟滋养层细胞发育进程，并结合体外基因表达调控技术、胚胎显微操作及相关动物模型，完整地揭示Galectin-1在滋养细胞增殖-分化进程中表达的时空特征、调控模式及相关重要分子间作用方式，并应用小鼠动物模型观察Galectin-1在胚胎着床和早期妊娠维持中的作用，为揭示着床失败和早期妊娠丢失机制、寻求相关治疗方法提供有价值的理论依据。

滋养细胞有序的增殖、分化在胚胎植入、胎盘形成及妊娠维持中起着重要的作用。子宫内膜上皮细胞是滋养细胞首先接触的母体细胞，两者对话及相互接受至关重要，一旦失衡会导致胚胎着床失败及早期妊娠丢失（EPL）。我们前期蛋白质组学研究发现，半乳糖凝集素-1（Gal-1）在人EPL 的滋养细胞中表达较正常对照显著降低，提示Gal-1 在早期胎盘发育、母胎对话中起重要作用。小鼠滋养层干细胞系（TSC）与体内的滋养层细胞谱系的基因表达模式相似，因此本项目应用模式动物和TSC模拟体内滋养细胞谱系早期发育过程研究Gal-1在胚胎着床中的作用及机制。我们的研究发现小鼠卵母细胞、原核期和着床前分裂期胚胎及囊胚（受精卵，2、4、8细胞和囊胚）均有Gal-1表达，且在囊胚期表达最高；Gal-1表达水平自TSC诱导分化即开始升高，第2-5天显著升高，之后下降；TSC分化过程中Gal-1启动子甲基化水平与Gal-1的表达水平呈负相关，推测 Gal-1在TSC分化过程中的有序表达受启动子甲基化的调控。运用滋养细胞-子宫内膜共培养体系模拟母胎界面，发现共培养后促进TSC的分化和侵袭，并上调TSC 的Gal-1表达，加入外源性Gal-1也能增强TSC分化和侵袭能力。提示子宫内膜上皮细胞能分泌某种因子上调滋养细胞Gal-1表达，从而促进其分化和侵袭。为了进一步验证Gal-1对滋养细胞侵袭能力的影响，我们应用SiRNA和腺病毒转染方式分别进行敲低和过表达TSC中的Gal-1，发现Gal-1过表达组TSC侵袭能力显著增高，Gal-1敲低组显著降低，提示Gal-1对滋养细胞侵袭和植入起到关键作用；为此，观察了SiRNA和腺病毒转染组EMT、MMPs、TGF-β通路中相关因子的变化，发现Gal-1通过对TGF-β信号通路相关下游基因EMT及MMPs表达的影响进而调控TSC的侵袭功能。本项目揭示了Gal-1 在胚胎早期发育各阶段和Gal-1及相关重要蛋白在滋养细胞增殖、分化及胎盘早期发育进程中的时空表达特征；明确了Gal-1通过TGF-β信号通路调控滋养细胞侵袭、并在胚胎着床过程母胎对话中发挥重要作用的机制，为改善胚胎着床及预防早期妊娠丢失提供了理论依据。