【作者】 宋宇鹏；

【导师】 庄洪兴；

【作者基本信息】 北京协和医学院 ， 整形外科， 2011， 博士

【摘要】 目的筛查先天性小耳畸形患者整个基因组存在甲基化水平异常的CpG岛和CpG位点及其相关差异基因,进一步检测差异基因的RNA表达水平,研究差异基因CpG岛的甲基化水平和基因表达的关系,同时检测去甲基化药物5氮杂胞苷干预后差异基因RNA表达水平的改变,进而探讨基因甲基化水平的异常与先天性小耳畸形发病之间的关系,为其发病机制提供新的科学理论。方法收集先天性小耳畸形患者的残耳软骨组织为研究对象,标记为实验组；非耳畸形患者的正常耳软骨组织作为正常对照,标记为对照组。选用基于甲基化DNA免疫共沉淀技术的Nimblegen CpG启动子芯片,对实验组3例及对照组3例样本的基因组DNA进行全基因组28226个CpG岛扫描,用高解析度芯片扫描仪检测杂交信号并输出影像,运用GenePix Pro6.0软件提取数据并输出excel表格。按照其中一组全部高甲基化而另一组全部低甲基化的标准筛选,确定两组之间存在CpG岛甲基化水平差异的基因。选用SpectroCHIP芯片,运用基质辅助激光解吸附电离飞行时间质谱分析仪对实验组47例,对照组31例的差异基因的CpG岛进行各个CpG位点的检测,输出的信号通过EpiTYPER软件进行分析并输出数据。对数据进行t检验,筛选出存在甲基化水平差异的CpG位点。选取实验组3例,对照组3例,在体外分离、培养残耳软骨细胞和正常耳软骨细胞,光镜下直接观察细胞形态变化,CCK-8法计数细胞,绘制细胞生长曲线,进行Ⅱ型胶原免疫组化染色。将对照组第3代细胞选取部分作为对照2组。对照2组加入不同浓度的5氮杂胞苷干预,通过CCK-8法绘制生长曲线和计算细胞活力评估此药物对细胞增殖的影响及细胞毒性检测。运用实时荧光定量PCR技术对实验组3例组织、1例细胞及对照组1例组织、1例细胞进行差异基因RNA表达水平的检测,同时检测对照2组2例细胞的差异基因RNA表达水平,对输出的Ct值应用Prizm 4统计分析软件进行T-test分析,找出实验组与对照组间RNA表达水平具有统计学差异的差异基因。同时对比对照组与对照2组间RNA表达水平具有统计学差异的差异基因。结果1.实验组与对照组在全基因组范围内,存在36个具有甲基化水平差异的CpG岛,其中有29个与已命名的29个基因相关,初步建立了先天性小耳畸形的甲基化谱。2.实验组与对照组在COL18A1、MYH14、RBMY1A1、ZIC3这四个差异基因的CpG岛内有6个CpG位点的甲基化水平存在差异且具有统计学意义。3.实验组与对照组的原代及1、2、3代软骨细胞在生物学性状及相关特异性上均无明显差异,细胞增殖及毒性实验证实5氮杂胞苷对正常耳软骨细胞的生长具有剂量依赖性的抑制作用。4.在软骨组织中,COL18A1基因的RNA表达水平在实验组较对照组低,具有非常显著的差异；MYH14、RBMYA1这2个基因的RNA表达水平在实验组均较对照组高,且具有显著性差异；ZIC3基因的RNA表达水平在实验组较对照组高,但不具有显著差异。在软骨细胞中,COL18A1, MYH14, ZIC3这3个基因的RNA表达水平在实验组均较对照组高,但不具有显著性差异；对照组与对照2组在COL18A1, MYH14、ZIC3这3个基因的RNA表达水平在对照2组均较对照组高,且具有显著性差异。结论先天性小耳畸形残耳软骨组织与正常耳软骨组织在全基因组DNA甲基化水平上有36个CpG岛存在差异,进一步对存在CpG岛甲基化水平差异的其中4个差异基因的具体CpG位点进行检测,发现4个差异基因共有6个CpG位点具有甲基化水平差异,对此4个差异基因的RNA表达水平检测发现5氮杂胞苷的去甲基化作用能够促进基因的表达,证实差异基因的表达水平与DNA甲基化相关；MYH14、RBMY1A1这2个差异基因在实验组和对照组的表达差异符合CpG岛甲基化水平的差异,表明在先天性小耳畸形中MYH14和RBMY1A1的表达调控与DNA的甲基化水平密切相关,从而初步推断出先天性小耳畸形的发病可能与DNA的甲基化水平有着密切的关系。更多还原

【Abstract】 PurposeScreening for abnormal methylation in CpG islands and CpG sites through whole genome of congenital microtia to identify their associated genes. RNA expression levels of these genes are also evaluated to study the relationship between CpG islands methylation level and gene expression. Meanwhile, the changes of gene expression after the intervention of 5-Azacytidine which is one of demethylation agent are detected.The study was designed to discuss the relationship between abnormal methylation level of genes and the etiology of congenital microtia.MethodTo collect the residual ear cartilage of microtia as the research object,marked as experimental group; normal ear cartilage of patients without ear malformations as control, marked as control group.Choosing Nimblegen CpG promoter array based on methylated DNA immunoprecipitation to screen the 28226 CpG islands in the whole genome of both experimental group and control group,the signals on the microarray were scanned by high-resolution Chip scanner.The data were extracted and exported to excel by GenePix Pro6.0 software.According to the standard that one group is hypermethylation and the other group is hypomethylation,the genes with differential methylated CpG islands were selected. Choosing SpectroCHIP array based on matrix-assisted laser desorption/ionization-time of flight mass spectrography analysis to detect the methylation level of each CpG site in abnormal methyletion CpG islands of both experimental group and control group.The signals were analyzed by EpiTYPER software and the data were exported to excel.Using t-test to analyse data between groups,the CpG sites with differential methylation level were elected. Collecting both experimental group and control group to isolate and culture chondrocytes in vitro,the morphologic changes were observed under light microscope,the amount of chondrocytes was evaluated by cell counting kit-8 in order to generate cell growth curves,and the type II collagen was tested by immunohistochemisty staining.Choosing part of control group chondrocytes as control group 2 to interfere the growth by 5-Azacytidine in different concentration,the influence of cellular proliferation and toxicity were evaluated by cell growth curves and cellular viability.Using the real-time quantitative PCR to test the RNA expression level of abnormal methylated genes between each group in ear cartilage and chondrocytes,the Ct values were analyzed by Prizm 4 software to select the abnormal methylated genes with differential RNA expression.Result1. There were thirty-six CpG islands with differential methylated level in whole genome between experimental group and control group,in which twenty-nine CpG islands were connected with twenty-nine named genes.The profile of DNA methylation patterns along the entire genome was initially established.2. In the abnormal methylated CpG islands of four differential genes including COL18A1,MYH14,RBMY1A1 and ZIC3, six differentially methylated CpG sites were found with statistical significance.3. The biological character and specificity showed no differences between each chondrocyte generation of experimental group and control group.Cell proliferation and toxicity test proved that the 5-Azacytidine has a dose dependent suppressive effect on the growth of the normal chondrocyte.4. In the cartilage, RNA expression in COL18A1 of experimental group was lower than that in control group with very significant difference. RNA expression in MYH14 and RBMYA1 of experimental group were both higher than that in control group with significant difference. RNA expression in ZIC3 of experimental group was higher than that in control group without significant difference.In chondrocyte, RNA expression in COL18A1,MYH14 and RBMY1A1 of experimental group were all higher than that in control group without significant difference. RNA expression in COL18A1,MYH14 and RBMY1A1 of control group 2 were all higher than that in control group with significant difference.ConclusionThere were thirty-six CpG islands with differential methylated level in whole genome between congenital microtia ear cartilage and normal ear cartilage. Screening for differential methylated CpG sites in four abnormal methylated CpG islands in genes,it is found that six CpG sites had differential methylation level between experimental group and control group. It is considered that the demethylation effect of 5-Azacytidine could promote gene expression,and proved that gene expression was associated with DNA methylation.The RNA expression of MYH14 and RBMY1A1 in experimental group and control group matched the difference of CpG islands methylation level,so it is a conclusion that the regulation of gene expression is tightly connected with DNA methylation in microtia.Therefore,the etiology of congenital microtia may have a close relationship with DNA methylation.更多还原