人肝细胞中FⅧ基因5'端CpG岛甲基化状态的研究

Study on CpG Island Methylation Status of FⅧ Gene in Hepatic Cells

本实验通过建立稳定的重亚硫酸盐测序(bisulfite-sequencing PCR,BSP)检测平台,研究人类永生化肝细胞LO2与人肝组织细胞中凝血因子Ⅷ(coagulation factorⅧ,FⅧ)基因5'端Cp G岛的甲基化状态及该Cp G岛的甲基化在FⅧ表达调节中的作用。运用RT-PCR、Western-blot检测FⅧ在LO2细胞与人肝组织中的转录及表达,Methprimer软件预测FⅧ基因5'端Cp G岛并设计BSP引物,BSP联合TA克隆及质粒PCR各验证十个阳性单克隆送测序,QUMA软件对测序结果进行甲基化位点分析,以(甲基化CG位点个数)/(甲基化CG位点个数+非甲基化位点个数)计算甲基化率。结果显示,新鲜分离的人肝组织细胞能够稳定表达FⅧ,LO2细胞不能表达;FⅧ基因5'端存在一个278 bp的Cp G岛,LO2细胞中该Cp G岛的甲基化率为93.48%,正常人肝组织中该Cp G岛的甲基化率为93.91%。本实验成功建立稳定的BSP检测平台,并发现在LO2细胞与人肝组织中,FⅧ基因5'端均存在一个被高度甲基化的Cp G岛,人肝组织中FⅧ基因5'端Cp G岛的甲基化可能参与维持体内FⅧ的低水平表达。

The objectives of this research were to establish stable bisulfite sequencing PCR(BSP) testing platform,detecting the methylation status of Cp G island in human immortalized hepatocyte LO2 and hepatic tissue,explore the regulation function of methylated Cp G island in F Ⅷ gene's expression. Using RT-PCR and Western-blot detected the F Ⅷ gene expression in LO2 cells and hepatic tissue.Methprimer software predicted the Cp G island and designed BSP primers, BSP combine TA clone detected the methylation status of the Cp G island in LO2 cells and hepatic tissue, and tested ten positive monoclone and sent it to a sequencing facility. QUMA software analysed the methylation site, and calculated the methylation rate of Cp G island using(methylated Cp G sites)/(methylated Cp G sites+unmethylated Cp G sites). RT-PCR and Western-blot showed that there is F Ⅷ express in normal liver cells but not in LO2 cells. We found a 278 bp Cp G island in the upstream sequence of FⅧ gene. The methylation status of Cp G island was 93.48% in hepatic tissue, 93.91% in LO2 cells. We established stable BSP testing platform and found there was a highly methylated Cp G island in the 5' end of F Ⅷ gene in human immortalized hepatocyte LO2 and hepatic tissue, we proposed that methylated Cp G island of the 5' end of FⅧ gene involved in maintaining FⅧ low level expression in hepatic tissue.