血管生成素家族调节人早孕滋养细胞浸润功能的分子机制研究

Trophoblast cells are invasive like tumor cells, Evidence indicates that a variety of factors could regulate invasion of trophoblast cells. However, the exact mechanism remains unclear. Our previous study showed the regulatory effect of first trimester human decidual stromal cell conditioned media (DSAM) on trophoblast cell invasion. In addition, we found a invasion-associated protein-Angiopoietin (Ang) in the decidual microenvironment which could regulate the trophoblast invasion by protein chips. In this study, we plan to investigate the molecular mechanism study of Angiogenin family in regulating trophoblast cell invasion: We first plan to validate the microarray results by ELISA and Immunoblotting analysis; Then different concentrations of Ang protein and blocking antibodies are added in the media of trophoblast cells, and the cell invasion will be investigated. In addition, the expression of bFGF, VEGF, EGF will be examined to clarify the potential paracrine and autocrine pathway, and clarify the signal pathway by investigating STAT3, ERK levels in the JAK-STAT, MAPK, PI3/AKT, and NF-kb pathways. Finally we plan to investigate the different expression of Ang in placentas of hydatidiform mole, choriocarcinoma, miscarriage and preeclampsia. Our study may clarify the pathogenesis mechanism of the trophoblastic diseases, and provide the oretical and experimental evidence for the diagnosis and treatment.

滋养细胞具有类似肿瘤细胞的浸润能力，研究表明滋养细胞的浸润过程受到多种因子的调控，但其确切机制仍不清楚。我们在前期研究中发现人早孕子宫蜕膜条件培养液可调节滋养细胞的浸润能力，并通过蛋白芯片筛选出了蜕膜微环境中调节滋养细胞浸润的相关蛋白-血管生成素（Ang）。本项目拟在前期研究基础上进一步阐明血管生成素家族调节滋养细胞浸润功能的分子机制：拟采用ELISA、免疫印迹等方法验证芯片结果；检测不同浓度的Ang蛋白及其封闭抗体对滋养细胞浸润功能的影响；将Ang蛋白与滋养细胞共培养，检测bFGF、VEGF、EGF等的表达变化以寻找潜在的自分泌、旁分泌通路，检测JAK-STAT、MAPK通路、PI3/AKT和NF-kb通路中STAT3、ERK等磷酸化水平，探寻相关的信号通路；检测Ang在葡萄胎、绒癌、流产绒毛及子痫前期患者胎盘中的表达差异。以期阐明滋养细胞相关疾病的发病机制，为其诊治提供理论和实验依据。

滋养细胞具有如同肿瘤细胞一样的浸润能力，但与肿瘤细胞不同的是，滋养细胞浸润是有节制的生理性浸润，浸润异常将导致病理性妊娠的发生。本课题组前期研究中发现，人早孕蜕膜基质细胞能影响滋养细胞的浸润功能，并通过蛋白芯片筛选出了滋养细胞浸润相关因子—angiogenin，推测angiogenin可能对滋养细胞浸润和迁移功能发挥重要调控作用，本项目拟检测其相关的研究机制。. 目的及方法：通过qPCR和Western blot方法明确Angiogenin在不同滋养细胞系和临床样本中的表达；在构建过表达质粒和siRNA的基础上，使用Transwel实验和划痕实验验证Angiogenin对滋养细胞浸润和迁移功能的调控作用；通过qPCR、Western blot、报告基因实验和功能挽救实验等方法阐明参与Angiogenin调控的可能miRNA机制；通过对正常患者和PE患者临床样本中Angiogenin等相关因子的相关性分析进一步论证Angiogenin及其相关miRNA机制在PE发病中的作用。. 结果：1. Angiogenin参与了滋养细胞浸润和迁移功能的调控，它可以明显促进滋养细胞的浸润和迁移功能，其作用依赖于调控下游MMP-2表达；2. miR-519d-3p可以抑制滋养细胞的浸润和迁移能力，主要依赖于调控靶基因MMP-2而发挥抑制作用；3. Angiogenin通过miR-519d-3p/MMP-2通路发挥促进滋养细胞浸润和迁移功能的作用，同时Angiogenin/miR-519d/MMP-2调控通路可能参与了临床PE的发病过程。