本文關(guān)鍵詞：N6AMT1多態(tài)性、葉酸對(duì)砷代謝影響及砷致DNA甲基化改變的研究　出處：《中山大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 砷代謝 N6AMT1 基因多態(tài)性 葉酸 DNA甲基化

【摘要】：砷是自然界中廣泛存在的一類致癌物,飲水型慢性砷暴露已經(jīng)成為威脅人類健康的全球性公共衛(wèi)生問(wèn)題。我國(guó)內(nèi)蒙古自治區(qū)的河套平原是世界上最嚴(yán)重的飲水型砷暴露地區(qū)之一。長(zhǎng)期慢性砷暴露可導(dǎo)致相關(guān)非癌癥疾病(如皮膚損傷、高血壓、糖尿病)和癌癥(如皮膚癌、腎癌、肺癌、膀胱癌)發(fā)生率和死亡率的上升。在人體內(nèi),無(wú)機(jī)砷(inorganic arsenic,iAs)主要由(+3價(jià)氧化態(tài))砷甲基轉(zhuǎn)移酶(arsenic(+3 oxidation state)methyltransferase,As3MT)催化生成一甲基砷酸化合物(monomethylarsonic acid,MMA)和二甲基砷酸化合物(dimethylarsinic acid,DMA),通過(guò)尿液排出體外。之前我們研究組利用酵母基因突變篩選系統(tǒng),發(fā)現(xiàn)N-6-腺嘌呤特異性DNA甲基轉(zhuǎn)移酶1(N6 adenine-specific DNA methyltransferase1,N6AMT1)參與砷的甲基化代謝,并通過(guò)體外細(xì)胞實(shí)驗(yàn)發(fā)現(xiàn)N6AMT1與As3MT在砷代謝過(guò)程中存在相互作用,N6AMT1是一種有潛力的新型砷甲基化轉(zhuǎn)移酶。許多研究證實(shí),遺傳多態(tài)性是導(dǎo)致個(gè)體之間對(duì)砷毒性和致癌性作用產(chǎn)生易感性差異的因素之一,但目前還沒(méi)有以中國(guó)人群為基礎(chǔ)評(píng)價(jià)N6AMT1基因多態(tài)性對(duì)砷代謝影響的研究。在本研究中,我們以289名生活在內(nèi)蒙古自治區(qū)河套平原五原縣農(nóng)村地區(qū)、受飲水型慢性砷暴露(水砷濃度㧐10μg/L)影響的居民為研究對(duì)象,運(yùn)用iPLEX Gold技術(shù)對(duì)挑選的N6AMT1基因和As3MT的基因多態(tài)性位點(diǎn)進(jìn)行分型檢測(cè),并以高效液相色譜-電感耦合等離子體質(zhì)譜檢測(cè)個(gè)體尿液中砷代謝產(chǎn)物含量,經(jīng)過(guò)數(shù)據(jù)統(tǒng)計(jì)分析,發(fā)現(xiàn)5個(gè)N6AMT1基因多態(tài)性位點(diǎn)(rs1003671、rs7282257、rs2065266、rs2738966、rs2248501)和N6AMT1基因單倍體型GGCCAT與砷代謝相關(guān),特別體現(xiàn)在%iAs的顯著差異上。N6AMT1基因單倍體型GGCCAT與As3MT基因單倍體型GCAC的組合,表現(xiàn)出砷代謝能力和效率的疊加效應(yīng),攜帶該單倍體型組合的個(gè)體有更強(qiáng)的砷代謝能力和效率。流行病學(xué)研究發(fā)現(xiàn),膳食營(yíng)養(yǎng)素(如葉酸、維生素B12、膽堿、蛋氨酸等)的攝入情況也會(huì)影響人體內(nèi)砷的代謝和基因組DNA甲基化水平,其中作為一碳循環(huán)底物的葉酸的影響尤為突出。但目前基于中國(guó)人群探討葉酸補(bǔ)充對(duì)砷體內(nèi)代謝情況及DNA甲基化影響的研究比較少。在本研究中,我們把研究對(duì)象(與基因多態(tài)性研究部分相同的群體)隨機(jī)分組成3組,進(jìn)行為期8周的雙盲、安慰劑對(duì)照葉酸補(bǔ)充試驗(yàn),經(jīng)過(guò)8周的葉酸補(bǔ)充,與安慰劑對(duì)照組相比,低劑量(400μg/d)和高劑量(800μg/d)葉酸補(bǔ)充組個(gè)體的血清葉酸水平明顯地升高,而血漿同型半胱氨酸水平則明顯地降低。葉酸補(bǔ)充組個(gè)體對(duì)無(wú)機(jī)砷甲基化代謝能力明顯提高,他們尿液中%iAs、%MMA更低而%DMA更高,而且高劑量組在各指標(biāo)上都表現(xiàn)更優(yōu)。此外,葉酸的補(bǔ)充對(duì)機(jī)體外周血全基因組DNA甲基化水平有提高的作用,提示其對(duì)維持砷暴露個(gè)體正常DNA甲基化水平有積極的意義。無(wú)機(jī)砷不會(huì)引起基因組的點(diǎn)突變,普遍認(rèn)為表觀遺傳改變?cè)跓o(wú)機(jī)砷致癌作用中擔(dān)當(dāng)重要角色,擾亂正常DNA甲基化模式會(huì)導(dǎo)致體內(nèi)原癌基因和抑癌基因的表達(dá)失控,從而誘發(fā)病變的發(fā)生。但是目前對(duì)于DNA甲基化模式紊亂與砷誘導(dǎo)癌癥發(fā)生的相關(guān)性,以及變化的甲基化模式在多代之間的保留情況研究得很少。在本研究中,我們以直系親屬3代人為基礎(chǔ),從內(nèi)蒙古自治區(qū)河套平原招募到代表砷暴露組、非砷暴露組和皮膚損傷病人組的119人,通過(guò)全基因組DNA甲基化水平分析和Illumina Infinium Human Methylation450微珠芯片甲基化位點(diǎn)檢測(cè),發(fā)現(xiàn)砷暴露組每一代個(gè)體的全基因組DNA甲基化水平都比非暴露組同代個(gè)體的低,皮膚損傷病人組的水平比暴露組和非暴露組所有的代數(shù)亞組都明顯要低。經(jīng)過(guò)分層分位數(shù)標(biāo)準(zhǔn)化法和Combat相結(jié)合的方式對(duì)甲基化數(shù)據(jù)進(jìn)行標(biāo)準(zhǔn)化和批次校正處理,暴露組和非暴露組的3代之間有超過(guò)5000個(gè)基因組位點(diǎn)的甲基化水平存在明顯差異(P值0.0001),第2代人甲基化顯著差異的位點(diǎn)數(shù)量最多,提示生命早期階段(胎兒期/幼年期)可能對(duì)砷誘導(dǎo)的甲基化改變更為敏感�；诒┞督M和非暴露組3代之間共同存在顯著甲基化差異的964個(gè)探針,進(jìn)行等級(jí)聚類分析,皮膚損傷病人的甲基化模式和砷暴露組個(gè)體更為相似,但與有砷暴露歷史但沒(méi)發(fā)病的個(gè)體又存在區(qū)別,而非暴露組個(gè)體的甲基化模式與皮膚損傷病人和暴露組則區(qū)別明顯。綜上所述,本研究發(fā)現(xiàn)在中國(guó)人群中N6AMT1基因多態(tài)性與砷的生物甲基化代謝存在聯(lián)系,并且與As3MT基因的特定單倍體型組合存在相互作用關(guān)系;葉酸的補(bǔ)充對(duì)砷的甲基化代謝和全基因組DNA甲基化水平的維持有積極的作用;砷暴露會(huì)導(dǎo)致DNA甲基化模式發(fā)生改變,這種改變?cè)诙啻g有一定的重疊性,而且與相關(guān)病變的發(fā)生存在關(guān)聯(lián)。這些發(fā)現(xiàn)可以為尋找人群對(duì)砷毒性和致癌作用的易感性差異標(biāo)志物,以及尋找一種經(jīng)濟(jì)、有效預(yù)防地方性砷中毒的途徑,提供一定的理論基礎(chǔ)和實(shí)踐依據(jù)。
[Abstract]:Arsenic is a kind of widely exists in the nature of carcinogen, has become a global public health threat to human health of chronic arsenic exposure. Drinking water in the Inner Mongolia Autonomous Region Hetao Plain is one of the regions exposed to arsenic in drinking water is the most serious in the world. Chronic arsenic exposure can lead to non cancer diseases (such as hypertension, skin damage. Diabetes) and cancer (such as skin cancer, renal carcinoma, lung cancer, bladder cancer incidence and mortality rise). In the human body, inorganic arsenic (inorganic arsenic, iAs (+3) is mainly composed of oxidation state) arsenic methyl transferase (arsenic (+3 oxidation state) methyltransferase, As3MT) catalyze the formation of methyl arsenate compounds (monomethylarsonic acid, MMA) and two methyl arsenate compound (Dimethylarsinic acid, DMA), excreted through the urine. Before we study group using yeast gene mutation screening system, N-6- gland Purine specific DNA methyltransferase 1 (N6 adenine-specific DNA methyltransferase1, N6AMT1) involved in the methylation of arsenic, and through in vitro experiments showed that N6AMT1 interacted with As3MT in arsenic metabolism process, N6AMT1 is a new type of arsenic methylation potential transferase. Many studies have confirmed that genetic polymorphism is the result one of the factors of arsenic toxicity and carcinogenicity of susceptibility differences between individuals, but there is no study on the effect evaluation of N6AMT1 gene polymorphism on arsenic metabolism in China population. In this study, I have to live in the countryside 289 Wuyuan County, the Inner Mongolia Autonomous Region Hetao plain area, affected by chronic arsenic exposure in drinking water (water the concentration of arsenic? 10 g/L) affected residents as the research object, using the polymorphism of iPLEX gene and As3MT N6AMT1 Gold technology to choose the type of detection, and to High performance liquid chromatography inductively coupled plasma mass spectrometry detection of individual urinary arsenic metabolites after statistical analysis of the data, found that 5 N6AMT1 gene polymorphisms (rs1003671, rs7282257, rs2065266, rs2738966, rs2248501) and N6AMT1 gene haplotype GGCCAT and arsenic metabolism, especially in the significant difference on%iAs gene.N6AMT1 single type GGCCAT and As3MT gene GCAC haplotype combinations showed additive effect of arsenic metabolism capability and efficiency, with the combination of the individual haplotype has stronger arsenic metabolism capability and efficiency. Epidemiological studies have found that dietary nutrients (such as folic acid, vitamin B12, choline, methionine) intake will affect the metabolism the body of arsenic and DNA methylation, which affect the carbon cycle as the substrate of folic acid is particularly prominent. But based on the current population of Chinese leaves Study on effect of arsenic metabolism and DNA methylation of acid is relatively small. In this study, we take the research object (and the study of gene polymorphism groups in the same part) were randomly divided into 3 groups, a 8 week double-blind, placebo-controlled trials of folic acid supplementation, after 8 weeks of folic acid. Compared with the placebo group, low dose (400 g/d) and high dose (800 g/d) received folic acid individual serum folic acid levels significantly increased, and plasma homocysteine levels were significantly decreased. Folic acid supplementation group on arsenic methylation capacity of individuals increased significantly,%iAs in their urine, lower%MMA while the%DMA is high, and the high dose group in each index are better performed. In addition, folic acid supplement on the peripheral blood genomic DNA methylation level enhanced the activity, suggesting that the individuals are often exposed to arsenic in maintaining DNA methylation level There is positive significance. Do not cause a point mutation in the genome of inorganic arsenic, is generally believed that epigenetic changes play an important role in arsenic carcinogenesis, disrupting the normal DNA methylation patterns can lead to loss of control in vivo expression of oncogenes and tumor suppressor genes, which induce the disease. But the correlation between cancer and disorder induced by arsenic DNA methylation patterns, and retention of methylation pattern changes in multiple generations between very little research. In this study, we take immediate family members of 3 generations of human based, from the Inner Mongolia Autonomous Region to recruit on behalf of the Hetao Plain arsenic exposed group, non arsenic exposed group and 119 patients with skin injury group, through the whole genome the level of DNA methylation analysis and Illumina Infinium Human Methylation450 bead chip methylation detection, found that arsenic exposed group of each individual generation of whole genome DNA methyl The level of all non exposed group with individual low skin injury patients than the level of the exposed group and the control group were all significantly lower algebraic subgroups. Through hierarchical quantile standardization method and Combat method of combining the methylation data standardization and batch correction processing, and exposure group non exposed group of the 3 generation have obvious differences between the methylation level of more than 5000 genomic loci (P = 0.0001), the number of loci of second generations of the most significant differences in methylation, suggesting that the early stages of life (infant / juvenile) on methylation changes induced by arsenic is more sensitive. 964 probe the exposed group and non exposed group between 3 generations together significant methylation differences were based on hierarchical clustering analysis, methylation patterns and patient skin damage exposed group of individuals is more similar, but with arsenic exposure history but not the incidence of the individual And there is a difference, and the non exposed group of individual methylation patterns and skin injury patients and exposed group was obvious difference. In summary, this study found that in Chinese crowd biological methylation N6AMT1 gene polymorphism and the presence of arsenic, and there is interaction between specific haplotype combinations with As3MT gene; folic acid supplement the arsenic methylation and DNA methylation level in maintenance has a positive effect; arsenic exposure can cause DNA methylation pattern changes, this change has certain overlaps in between generations, are connected and related lesions. These findings can find differences on susceptibility to arsenic toxicity and the carcinogenic effect of markers, and find an economic, effective prevention of endemic arsenic poisoning and provide a theoretical basis and practical basis.

【學(xué)位授予單位】：中山大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R599.1

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本文編號(hào)：1433308