探究中国脑胶质瘤患者MGMT基因启动子区域甲基化与表达的关系

发布时间：2018-07-11 16:43

本文选题：CpG岛甲基化 + 脑胶质瘤　；参考：《西北大学》2014年硕士论文

【摘要】：脑胶质瘤(glioma)是成年人神经系统中最常见的肿瘤,死亡率居高。脑胶质瘤的治疗是世界公认的难题。烷基类化合物——替莫唑胺(temozolomid, TMZ)是一种新型咪唑四嗪类药物,对人类肿瘤细胞系具有广谱抗肿瘤活性。目前,美国和欧洲医学界已经将替莫唑胺定为治疗恶性脑瘤的“金标准”,国际医学界也已将替莫唑胺作为治疗恶性脑瘤的一线药物。 MGMT全称为O6-甲基鸟嘌呤-DNA甲基转移酶(O6-methylguanine-DNA methyltransferese),它是一种高效的DNA甲基转移酶,此酶可以将DNA分子上的甲基转移到自身氨基酸残基上,以修复各种烷化剂药物造成的细胞DNA烷基化损伤,特别是DNA分子中鸟嘌呤第6位氧原子上的甲基乃至烷基化损伤,从而有效的修复DNA损伤,防止细胞癌变和死亡。另外在脑胶质瘤患者的治疗过程中,MGMT蛋白过量表达会对化疗药物替莫唑胺产生耐药性,而MGMT基因启动子高甲基化,导致该基因转录水平下降(基因沉默),进而导致细胞内MGMT蛋白表达降低。因此,MGMT甲基化修饰作为预测替莫唑胺敏感性的分子标记物被广泛使用。目前临床上对于脑胶质瘤个体化用药指导的分子检测对象一直采用MGMT基因135,137等位点甲基化修饰的检测。但是,后期多项临床研究发现MGMT基因135,137位点的甲基化与药物敏感性没有显著相关性,这就会导致临床用药预测不准确。因此,寻找更准确的甲基化位点指导临床用药至关重要。本文采用了一种全新的基因甲基化检测技术——焦磷酸测序,建立了MGMT基因启动子区域全部甲基化位点定量检测的方法,并对15例脑胶质瘤样本和正常人的293细胞系进行MGMT基因启动子全部甲基化位点的定量检测,通过统计学检验发现了35个有显著性差异的CpG位点,后期通过实时定量PCR的方法分析脑胶质瘤病人样本MGMT基因的表达,然后将甲基化位点和表达进行关联分析,初步找到了8个与表达关系密切的CpG位点(P0.0001),为后期指导肿瘤个体化治疗有着重要的作用。焦磷酸测序技术是检测甲基化位点的“金标准”,其灵敏度高,周期短,一次检测可实现对单个CpG位点或者多个连续位点进行定量分析。
[Abstract]:Glioma (glioma) is the most common tumor in adult nervous system with high mortality. The treatment of glioma is recognized as a difficult problem in the world. Temozolomide (TMZ), an alkyl compound, is a novel imidazolium tetrazine drug with broad-spectrum antitumor activity against human tumor cell lines. Currently, the United States and European medical circles have set temozolamide as the "gold standard" for the treatment of malignant brain tumors. The international medical community has also used temozolomide as a first-line drug for the treatment of malignant brain tumors.MGMT is known as O6-methylguanine-DNA methyltransferase (O6-methylguanine-DNA methyltransferese), which is a highly effective DNA-methyltransferase. The enzyme can transfer the methyl from DNA molecule to its amino acid residues to repair the DNA alkylation damage caused by various alkylating agents, especially the methyl and even alkylation damage on the sixth oxygen atom of guanine in DNA molecule. Thus effectively repair DNA damage, prevent cell cancer and death. In addition, overexpression of MGMT protein in glioma patients may result in resistance to the chemotherapeutic drug temozolamide, while the promoter of MGMT gene is hypermethylated. The transcription level of the gene decreased (gene silencing) and the expression of MGMT protein decreased. Therefore, MGMT methylation modification is widely used as a molecular marker to predict the sensitivity of temozolamine. At present, methylation modification of 135137 locus of MGMT gene has been used for molecular detection of brain glioma guided by individualized drug use. However, there is no significant correlation between methylation and drug sensitivity in 135137 locus of MGMT gene, which leads to inaccurate prediction of clinical drug use. Therefore, it is important to find more accurate methylation sites to guide clinical drug use. In this paper, a novel gene methylation detection technique, pyrosequencing, was used to quantitatively detect all methylation sites in the promoter region of MGMT gene. All methylation sites of MGMT gene promoter were quantitatively detected in 15 glioma samples and 293 normal cell lines. 35 CpG loci with significant difference were found by statistical test. The expression of MGMT gene in glioma patients was analyzed by real-time quantitative PCR, and the methylation site and expression were analyzed by association analysis. Eight CpG loci (P0.0001) which were closely related to the expression of tumor were preliminarily identified, which play an important role in guiding individualized treatment of tumor at later stage. Pyrosequencing is a "gold standard" for the detection of methylation sites, which has high sensitivity and short period. A single test can be used to quantitatively analyze a single CpG locus or multiple consecutive sites.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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