【作者】 钟克力；

【导师】 王春友； 周汉新；

【作者基本信息】 华中科技大学 ， 普通外科， 2009， 博士

【摘要】 目的研究表观遗传修饰在胃癌发病机制中的作用。方法收集我科2007.10～2008.01病理确诊的8例胃癌患者肿瘤组织和正常胃粘膜组织，采用染色质免疫共沉淀联合芯片技术(ChIP-chip)在细胞全基因组范围内对两种组织细胞内组蛋白H3K27位点三甲基化水平进行高通量的检测，用染色质免疫共沉淀-实时定量聚合酶链反应(ChIP-qPCR)验证芯片检测结果，采用荧光定量反转录聚合酶链反应(qRT-PCR)检测两种组织细胞H3K27me3水平存在显著差异的目的基因mRNA表达水平，最后用甲基化DNA免疫沉淀-定量PCR(MeDIP-qPCR)方法检测目的基因启动子区域DNA甲基化水平。结果和正常胃粘膜组织细胞相比较，胃癌肿瘤细胞全基因组范围共筛选出227个基因组蛋白H3K27me3水平存在显著差异，其中有67个基因显示组蛋白H3K27三甲基化水平增高，160个基因组蛋白H3K27三甲基化水平降低；ChIP-qPCR技术检测两种组织细胞挑选基因组蛋白H3K27三甲基化水平的差异与CpG岛芯片检测结果一致，两种组织细胞挑选基因的mRNA表达水平和DNA甲基化水平均存在显著差异。结论与正常胃粘膜细胞相比，胃癌肿瘤细胞多个基因组蛋白H3K27三甲基化水平存在显著改变；基因组蛋白H3K27三甲基化对调控基因表达发挥沉默效应；挑选基因DNA甲基化水平亦存在明显变化，挑选基因DNA甲基化和组蛋白H3K27me3两种表观修饰方式存在相互作用，DNA甲基化修饰作用可能存在优势；表观遗传修饰在胃癌发病机制中发挥重要作用。ChIP-chip技术有利于进一步揭示胃癌发生的分子机制，发现新的治疗靶点。更多还原

【Abstract】 Objective To Study the effect of epigenetic gene modification on gastric cancer moleculor mechanism. Methods 8 cases including tumor tissue and gastric mucosa tissue in gastric cancer patients were collected from 10.2007 to 01.2008. For the first time chromatin immunoprecipitation linked to microarrays (ChIP-chip) was adopted to profile the variations in histone H3K27me3 of Genome-wide in tumor tissue and gastric mucosa tissue. ChIP-qPCR was used to validate the microrray results. mRNA Expression analysis of 3 selected genes by qRT-PCR was performed to confirm the correlations between H3K27me3 and gene expression. Finally, DNA methylation of five selected genes was detected with MeDIP-qPCR. Results 227(67 increased and 160 decreased) gene displayed significant histone H3K27me3 difference were found in gastric cancer cell compared with gastric mucosa tissue. The results of ChIP-qPCR were coincided well with ChIP-chip. There was marked difference between tumor tissue and gastric mucosa tissue about mRNA expression of selected genens, so is DNA methylation. Conclusions Compared with gastric mucosa tissue, there are significant changes in tumor tissue about histone H3K27me3 profiling and DNA methylation level. It is feasble that ChIP-chip technique detects histone H3K27me3 level. Histone H3K27me3 probably bring gene expression about "silence effect". There is interaction between histone H3K27me3 and DNA methylation. But DNA methylation is probably more important than H3K27me3. Epigenetic gene modification probably plays an important role in gastric cancer moleculor mechanism. These novel candidate genes may be become potential biomarkers or future therapeutic targets. The ChIP-chip technique will help further reveal gastric cancer molecular mechanisms and discover new therapeutic targets.更多还原