【摘要】：背景與目的：肺癌是復(fù)雜性疾病,由遺傳和環(huán)境因素共同作用所致。DNA甲基化體現(xiàn)了機(jī)體內(nèi)部遺傳和外界環(huán)境的交互作用,是一種早期事件,先于肺癌表型出現(xiàn)之前,在早期診斷中具有潛在的應(yīng)用價(jià)值。同時(shí),大部分肺癌患者在臨床確診之時(shí)已到中晚期,尋找一種有效的生物標(biāo)志物迫在眉睫。而液體活檢技術(shù)在腫瘤早期診斷、預(yù)后,以及精準(zhǔn)醫(yī)療中占有越來越重要的地位。另外,云南多個(gè)地區(qū)(如宣威)是世界著名的肺癌高發(fā)區(qū)。因此,本文旨在通過對(duì)該地區(qū)肺癌患者樣本進(jìn)行研究,探討血漿游離DNA(cell-free DNA,cfDNA)中多基因甲基化聯(lián)合檢測在肺癌早期診斷中的意義,并為云南肺癌發(fā)生機(jī)理提供參考依據(jù)。方法：本文研究對(duì)象來源于云南地區(qū),其中,23例樣本取自于肺癌患者腫瘤組織,42例樣本為肺癌患者血漿,此外,10例健康血漿樣本作為對(duì)照。對(duì)于所有樣本,均使用試劑盒提取DNA/cfDNA與重亞硫酸鹽處理,之后采用巢式甲基化特異性PCR(nested methylation-specific PCR, nMSP)法進(jìn)行甲基化檢測。首先根據(jù)文獻(xiàn)報(bào)道,在肺癌組織樣本中檢測了8個(gè)基因(p16、DLECI、 CDH1、 DAPK、RUNX3、APC、WIF1及MGMT)的甲基化情況,之后篩選出陽性率較高的基因,用于在血漿cfDNA中進(jìn)行檢測。由于9例患者既包括血漿,也含有組織,也對(duì)甲基化情況進(jìn)行一致性分析。此外,部分血漿樣本的甲基化及非甲基化產(chǎn)物,采用亞硫酸氫鹽測序法進(jìn)行驗(yàn)證。最后,分析甲基化與患者臨床病理信息之間的關(guān)系。結(jié)果：在組織樣本中,除MGMT以外的其余7個(gè)基因,其甲基化陽性率在39%-74%之間,并且聯(lián)合檢測時(shí)所有樣本至少1個(gè)基因發(fā)生甲基化的檢出率為96%,至少2個(gè)基因共同甲基化的檢出率為91%。其中,p16、DLEC、CDH1、 DAPK、RUNX3這5個(gè)基因的甲基化率較高(≥48%),因而在血漿cfDNA中進(jìn)行檢測。結(jié)果發(fā)現(xiàn),cfDNA中5個(gè)基因的甲基化率在14%-76%之間,并且聯(lián)合檢測時(shí)所有樣本至少1個(gè)或2個(gè)基因發(fā)生甲基化的檢出率分別為95%和71%。健康對(duì)照實(shí)驗(yàn)中,這5個(gè)基因的甲基化均無檢出。此外,9例配對(duì)血漿和組織樣本中的甲基化情況基本一致。結(jié)合臨床數(shù)據(jù)統(tǒng)計(jì)分析顯示,CDH1基因甲基化與肺癌病理分型、臨床分期和遠(yuǎn)處轉(zhuǎn)移有關(guān),而W1F1基因甲基化與肺癌臨床病理分型有關(guān)。最后,在組織或血漿DNA甲基化情況中,CDH1、RUNX3和W1F1基因易受患者年齡、性別或吸煙等因素的影響。主要結(jié)論：(1)云南地區(qū)肺癌發(fā)生發(fā)展與DNA甲基化密切相關(guān),多個(gè)基因的甲基化聯(lián)合檢測比單基因更具優(yōu)勢。(2)肺癌樣本血漿cfDNA中p16、DLECI、 CDH1、 DAPK、RUNX3等5個(gè)基因的甲基化檢出具有較高的敏感性和特異性,其組合可進(jìn)一步研究,有望開發(fā)成為云南肺癌早期診斷的表觀遺傳生物標(biāo)志物。(3)CDH1基因的甲基化與肺癌臨床多個(gè)病理特征相關(guān),在肺癌的預(yù)后判斷中具有一定的應(yīng)用價(jià)值。
[Abstract]:Background & objective: lung cancer is a complex disease. DNA methylation caused by genetic and environmental factors reflects the interaction between internal genetics and external environment. It is an early event before the appearance of lung cancer phenotype. It has potential application value in early diagnosis. At the same time, most lung cancer patients have reached the middle and late stage of clinical diagnosis, so it is urgent to find an effective biomarker. Fluid biopsy plays a more and more important role in early diagnosis, prognosis and accurate medicine. In addition, many areas of Yunnan Province (such as Xuanwei) are the world famous lung cancer high incidence area. Therefore, the purpose of this study was to investigate the significance of polygenic methylation in plasma free DNA (cell-free DNA cf DNA) in the early diagnosis of lung cancer, and to provide a reference for the pathogenesis of lung cancer in Yunnan Province. Methods: in this study, 23 samples were taken from tumor tissue of lung cancer patients, 42 samples were plasma samples from lung cancer patients, and 10 healthy plasma samples were taken as control. For all samples, DNA/cfDNA was extracted by kit and treated with sulfite, and then methylation was detected by nested methylation specific PCR (nested methylation-specific PCR, nMSP). Firstly, the methylation of eight genes (p16DLECI, CDH1, DAPKUX3RUNX3APCWIF1 and MGMT) were detected in lung cancer tissue samples according to literature reports, and then the genes with high positive rate were screened for detection in plasma cfDNA. Since 9 patients included both plasma and tissue, a consistent analysis of methylation was carried out. In addition, the methylation and demethylation products of some plasma samples were verified by bisulfite sequencing. Finally, the relationship between methylation and clinicopathological information was analyzed. Results: in tissue samples, the methylation positive rate of 7 genes except MGMT ranged from 39% to 74%. The detection rate of methylation of at least one gene in all samples was 96, and that of common methylation of at least two genes was 91. The methylation rate (鈮，

本文編號(hào)：2191191