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  本文選題：CpG島甲基化 + 腦膠質(zhì)瘤�。� 參考：《西北大學》2014年碩士論文

【摘要】：腦膠質(zhì)瘤(glioma)是成年人神經(jīng)系統(tǒng)中最常見的腫瘤,死亡率居高。腦膠質(zhì)瘤的治療是世界公認的難題。烷基類化合物——替莫唑胺(temozolomid, TMZ)是一種新型咪唑四嗪類藥物,對人類腫瘤細胞系具有廣譜抗腫瘤活性。目前,美國和歐洲醫(yī)學界已經(jīng)將替莫唑胺定為治療惡性腦瘤的“金標準”,國際醫(yī)學界也已將替莫唑胺作為治療惡性腦瘤的一線藥物。 MGMT全稱為O6-甲基鳥嘌呤-DNA甲基轉(zhuǎn)移酶(O6-methylguanine-DNA methyltransferese),它是一種高效的DNA甲基轉(zhuǎn)移酶,此酶可以將DNA分子上的甲基轉(zhuǎn)移到自身氨基酸殘基上,以修復各種烷化劑藥物造成的細胞DNA烷基化損傷,特別是DNA分子中鳥嘌呤第6位氧原子上的甲基乃至烷基化損傷,從而有效的修復DNA損傷,防止細胞癌變和死亡。另外在腦膠質(zhì)瘤患者的治療過程中,MGMT蛋白過量表達會對化療藥物替莫唑胺產(chǎn)生耐藥性,而MGMT基因啟動子高甲基化,導致該基因轉(zhuǎn)錄水平下降(基因沉默),進而導致細胞內(nèi)MGMT蛋白表達降低。因此,MGMT甲基化修飾作為預測替莫唑胺敏感性的分子標記物被廣泛使用。 目前臨床上對于腦膠質(zhì)瘤個體化用藥指導的分子檢測對象一直采用MGMT基因135,137等位點甲基化修飾的檢測。但是,后期多項臨床研究發(fā)現(xiàn)MGMT基因135,137位點的甲基化與藥物敏感性沒有顯著相關性,這就會導致臨床用藥預測不準確。因此,尋找更準確的甲基化位點指導臨床用藥至關重要。 本文采用了一種全新的基因甲基化檢測技術——焦磷酸測序,建立了MGMT基因啟動子區(qū)域全部甲基化位點定量檢測的方法,并對15例腦膠質(zhì)瘤樣本和正常人的293細胞系進行MGMT基因啟動子全部甲基化位點的定量檢測,通過統(tǒng)計學檢驗發(fā)現(xiàn)了35個有顯著性差異的CpG位點,后期通過實時定量PCR的方法分析腦膠質(zhì)瘤病人樣本MGMT基因的表達,然后將甲基化位點和表達進行關聯(lián)分析,初步找到了8個與表達關系密切的CpG位點(P0.0001),為后期指導腫瘤個體化治療有著重要的作用。 焦磷酸測序技術是檢測甲基化位點的“金標準”,其靈敏度高,周期短,一次檢測可實現(xiàn)對單個CpG位點或者多個連續(xù)位點進行定量分析。
[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.
【學位授予單位】：西北大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R739.41

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本文編號：2115913