【作者】 李明岳；

【导师】 王春友； 余小舫；

【作者基本信息】 华中科技大学 ， 普通外科， 2010， 博士

【摘要】 目的研究表观遗传修饰在胰岛分化及其相关基因(Pdx-1、Pax4、MafA和Nkx6.1)转录表达的作用。方法培养及收集小鼠胚胎干细胞(mES)(1×107)、小鼠成纤维细胞株NIH3T3细胞(1×107)和小鼠胰岛素瘤细胞株NIT-1细胞(1×107)三种细胞,采用甲基化DNA免疫共沉淀-实时定量PCR法(MeDIP-qPCR)检测3种细胞中与胰岛分化相关的基因、0ct4基因和MLH1基因的转录起始区DNA甲基化水平,采用染色质免疫共沉淀-实时定量PCR法(ChIP-qPCR)检测上述三种细胞各基因转录起始区组蛋白修饰(H3乙酰化、H3K4m3和H3K9m3修饰)的状况,同时采用实时定量RT-PCR检测上述三种细胞各基因mRNA表达水平,分析DNA甲基化、H3乙酰化、H3K4m3和H3K9m3修饰改变与基因表达之间的关系。结果(1)NIH3T3细胞Pdx-1、MafA和Nkx6.1基因转录起始区呈高甲基化,与mES和NIT-1细胞相比均明显增高(P<0.05),与基因表达存在负相关；(2)NIT-1细胞中Pdx-1、Pax4、MafA、Nkx6.1等基因转录起始区的H3K4m的修饰水平较mES和NIH3T3细胞明显增高(P<0.05),基因表达。(3)NIH3T3细胞中Pdx-1、Pax4、MafA、Nkx6.1等基因转录起始区的H3K9m3的修饰水平较mES和NIT-1细胞明显增高(P<0.05),基因不表达。结论组蛋白修饰(H3K4m3和H3K9m3)及DNA甲基化能相互协调,共同参与了Pdx-1、MafA、Nkx6.1和Pax4等与胰岛分化相关的基因的表达调控,对胚胎干细胞向胰岛分化具有重要的意义。更多还原

【Abstract】 Objective:To study the effect of epigenetic modification on the differentiation of islet cells and the expression of associated genes (Pdx-1、Pax4、MafA、Nkx6.1,etc). Method:Mouse embryonic stem cells(1×107), NIH3T3 cells(1×107) and NIT-1 cells(1×107) were cultured and collected. The promoter methylation status of islet differentiation-associated genes (Pdx-1,Pax4,MafA and Nkx6.1),Oct4 and MLH1 genes among different cell types were measured by using methylated DNA immunoprecipitation-real time quantitative PCR methods (MeDIP-qPCR). The promoter histone modification status of these genes among different cell types were measured by using chromatin immunoprecipitation-real time quantitative PCR methods. The expression of these genes in the three cell lines were measured by realtime quantitative PCR. Analyze the relationship between the differences of epigenetic modification(DNA methylation, H3 acetylation, H3K4m3 and H3K9m3) and gene expressions. Results:(1) In NIH3T3 cell, the transcription-initiation-site of Pdx-1, MafA and Nkx6.1 are high extent of methylaion status. Compared with mES cell and NIT-1 cell, NIH3T3 cell has a sigificantly higher level of DNA methylation modification (P<0.05). There are the Pearson correlation between expressions of Pdx-1、MafA、Nkx6 genes and methylation modification.(2) Compared with mES cell and with NIH3T3 cell, NIT-1 cell has a sigificantly higher level of H3K4m3 modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes (p<0.05), with sigificant level of gene expression. (3) Compared with mES cell and with NIT-1 cell, NIH3T3 cell has a sigificantly higher level of H3K9m3 modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes (p<0.05), with no gene expression. Conclusion:Histone modification (H3K4m3 and H3K9m3) and DNA methylation can regulate one another, and synergtically regulate the expression of islet differentiation-associated genes. These epigenetic modifications pose significant importance in the differentiation process from embryonic stem cell into islet cell.更多还原