反式脂肪酸对胎盘滋养层细胞迁移的影响及其机制研究

Intake of trans fatty acids (TFA) is associated with a higher risk of many diseases. It is reported that TFA were incorporated in high concentrations in placenta. In preterm infants and healthy term babies, trans fatty acids have been inversely correlated to infantile birth weight. However，the effect of trans fatty acids on early placental development remains unclear. The remarkable process of trophoblast cell migration is essential for early placental development and pregnancy success. This project will focus on the effect and its mechanism of trans fatty acids on the migration of human trophoblast cell through wingless (Wnt)/β-catenin signaling pathway. The contents include: Analysis of the effect of TFA on the migration of trophoblast cell using collagen and transwell in vitro migration models; Determination of the pivotal matrix metalloproteinases (MMPs) in the process of trophoblast cell migration modulated by TFA through Realtime PCR, ELISA, gelatin zymography and MMP overexpression; Analysis of the mechanism of TFA involved with Wnt/β-catenin signaling pathway in multiple levels, using recombinant Wnt 3a, glycogen synthase kinase (GSK)-3β inhibitor Li+, posttranscriptional gene silencing of Low-density lipoprotin receptor related (LRP) coreceptor, Wnt ligands soluble inhibitor Dickkopf-1 (Dkk1), combined with western blot analysis of phosphorylation of GSK-3β and β-catenin, immunofluorescence analysis of β-catenin distribution in cell cytoplasma and nucleus; Correlation analysis of TFA intake and abnormal early placenta development and its mechanism in vivo, by gas chromatography analysis of TFA content in human early pregnant maternal blood and placental villi, histological staining, immunohistochemistry and western blot. We will provide evidence of potential influences for TFA in early placental development and TFA intake quantity limit of early pregnant women in this project.

反式脂肪酸（TFA）的摄入与许多疾病有关。已发现TFA 可在胎盘组织大量蓄积，且与新生儿体重负相关，但其对胎盘发育的影响尚未阐明。滋养层细胞迁移对胎盘早期发育至关重要。本项目对TFA通过Wnt/β-catenin信号通路影响人滋养层细胞（TC）迁移的作用及其机制进行基础研究。具体内容包括：应用体外迁移模型，分析TFA对TC迁移的影响；应用荧光定量PCR、酶联免疫吸附法、酶谱法和基因过表达，分析参与TFA影响TC迁移过程的关键酶MMPs；应用重组Wnt 3a、GSK-3β抑制剂、LRP基因沉默和Wnt 3a竞争性抑制剂，从多个层面分析该通路参与TFA影响TC迁移的机制；应用气相色谱法分析人早期妊娠母体血液及胎盘绒毛组织TFA含量，结合组织学染色和免疫组化分析其与胎盘早期发育异常的相关性及作用机制。本项目旨在为TFA对胎盘早期发育的影响及早期妊娠人群TFA摄入限量的研究提供科学依据。

研究表明，食品加工过程产生的反式脂肪酸的摄入与许多疾病有关。已发现反式脂肪酸可以在胎盘组织大量蓄积，且与新生儿体重负相关，但其对胎盘发育的影响尚未阐明。滋养层细胞的迁移直接影响胎盘的形成，是胎盘发育至关重要的环节。因此，本论文研究了食品加工过程中产生的两种主要反式脂肪酸，反-9-十八碳烯酸（反油酸）和反-9，反-12-十八碳二烯酸（反亚油酸）对滋养层细胞迁移的影响，并对其作用机制进行了探讨。.通过体外迁移模型，发现反油酸和反亚油酸能够明显抑制滋养层细胞的体外迁移，其作用呈现明显的时间依赖性和剂量依赖性。进一步的分析发现反油酸和反亚油酸能够明显抑制其在调节滋养层细胞迁移能力中发挥关键作用的基质金属蛋白酶MMP-2的表达，并证明了其在反式脂肪酸调节滋养层细胞迁移能力中作用。分析了反式脂肪酸影响滋养层细胞体外迁移过程中Wnt/β-catenin 信号通路的活化状态，发现反式脂肪酸能够抑制该通路的活化状态。进一步应用拮抗实验分析、证明了Wnt/β-catenin 信号通路参与反式脂肪酸影响滋养层细胞体外迁移的作用机制。分析了早期妊娠绒毛组织及母体血液中反式脂肪酸等多种脂肪酸异构体的组成和含量，发现了胎盘早期发育异常绒毛组织特异脂肪酸异构体的组成及含量的显著变化。.综上，本研究分析了反式脂肪酸对滋养层细胞体外迁移的影响及其作用机制，为开拓反式脂肪酸对胎盘早期发育影响的认识提供了科学依据。