Cell and Molecular mechanisms of placental syncytialisation : implications for the pathogenesis of pre-eclampsia

(1)用细胞合胞化模型研究了胎盘滋养层细胞合胞化的独特生物学过程(细胞滋养层细胞系BeWo跟随Forsklin增加细胞内cAMP)。(2)获得了稳定表达融合蛋白绿色荧光蛋白和人组蛋白H2B或红色荧光蛋白融合蛋白和人细胞色素c氧化酶VIII亚基的线粒体靶向序列的BeWo细胞系。利用这两个表达不同颜色荧光蛋白的BeWo细胞系进行了细胞融合(合胞化)的可视化，并建立了基于荧光激活细胞分选的定量测定合胞化率的方法。(3)利用DNA微阵列在合胞化的细胞模型中研究了合胞化的时间过程。在Forsklin治疗后表达增加的基因中，许多基因是细胞通讯和新陈代谢所必需的。已知的几个参与细胞黏附和融合的基因在Forsklin暴露后非常早的时候就显著改变了表达水平，从而先于BeWo细胞的形态融合。进一步分析这些数据和总体上的表达谱将有助于理解胎盘合体滋养层细胞形成的功能基础。(4)CD98表面抗原在调节合胞化和氨基酸运输活性方面的生理重要性已被平行研究，无论是使用反义寡核苷酸还是RNA干扰。结果表明，CD98参与了合体滋养层细胞形成所必需的细胞融合过程，在这一重要生理事件中，氨基酸转运活性也通过该膜蛋白的表达来调节。

(1)A unique biological process of placental trophoblast syncytialisation has been studied using a cell model of syncytialisation (the cytotrophoblast cell line BeWo following increased intracellular cAMP by forskolin).(2)Stable BeWo cell lines constitutively expressing either a fusion protein green fluorescent protein and human histone H2B or a fusion protein of red fluorescent protein and the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase have been generated. Using these two BeWo cell lines expressing fluorescent protein of different colour cell fusion (syncytialisation) has been visualized and a fluorescence activated cell sorting based assay for determining quantitatively the rate of syncytialisation has been developed.(3)The time course of syncytialisation has been studied using DNA microarray in a cell model of syncytialisation. Among the genes with increased expression following forskolin treatment were many required for cellular communication and metabolism. Several genes known to be involved in cell adhesion and fusion have markedly changed expression levels very early following forskolin exposure, thus preceding morphological fusion of BeWo cells. Further analysis of this data and expression profiling in general will be able to contribute to understanding the functional basis for the formation of the placental syncytiotrophoblast.(4)The physiological importance of CD98 surface antigen in regulating syncytialisation and amino acid transport activity has been studied in parallel using either antisense oligonucleotides or RNA interference. The results show that CD98 is involved in the process of cell fusion necessary for syncytiotrophoblast formation and that during this physiologically important event amino acid transport activity is also regulated through expression of this membrane protein.