Regulatory mechanism to tissue factor gene expression by its methylation/demethylation

组织因子（TF）是一种启动血液凝固的因子VII/VIIa受体。TF被认为具有其他功能，如参与胚胎发生、血管生成、肿瘤转移和/或信号转导。TF基因的表达受AP-1、kappaB、SP-1和erg1的几个is元件以及结合这些顺式元件的转录因子的调控。由于在胞嘧啶残基上存在大量的CG岛特异性甲基化位点，因此预计会有另一种对TF基因表达的调控机制。在这项研究中，我们通过对TF基因启动子区域的甲基化和/或去甲基化来研究其表达的可能调控机制。我们还研究了几种细胞的分化和去分化/转化与TF基因甲基化/去甲基化之间的关系。我们建立了一种竞争性的RT-PCR，可以使用特定的引物集定量测量几种正常或转化细胞中表达的TF m-RNA。我们还建立了灵敏的三明治酶联免疫吸附法来测量这些细胞中产生的TF抗原。在正常血细胞中，单核细胞和粒细胞表达TF，而淋巴细胞不表达。TF基因在猴雄性生殖组织中的表达存在显著差异。精囊特异性表达非常高水平的TF-mRNA。这些结果表明，在白细胞从共同前体和组织推测分化过程中，TF基因的表达可能受到甲基化/去甲基化的调节。

Tissue Factor (TF) is a factor VII/VIIa receptor that initiates blood coagulation. TF is suggested to have other functions such as involvement in embryogenesis, angiogenesis, tumor metastasis, and/or signal transdution. TF gene expression is regulated by several is-elements of AP-1, kappaB, SP-1 and Erg-1 and transcriptional factors to bind these cis-elements. Another regulatory machinery to TF gene expression is expected because there are a lot of CG island specific site for methylation at cytosine residues.In this studies we examined a possible regulation mechanism of TF gene expression through methylation and/or demethylation of its promoter region. We also investigated a relationship between differentiation and de-differentiation/transformation of several cells and methylation/ demethylation of TF gene.We established a competitive RT-PCR which is enable to quantitative measurement of TF m-RNA expressed in several normal or transformed cells using specific primer sets. We also established sensitive sandwich enzyme-linked immuno-sorbent assay to measure TF antigen produced in these cells.In normal blood cells monocytes and granulocytes expressed TF but lymphocytes did not. Marked different expression of TF gene in monkey male reproductive tissues. Seminal vesicle specifically expressed very high level of TF-mRNA.These suggest TF gene expression should regulated by methylation/demethylation during differentiation of leukocytes from common precursor and tissue speculation.