【摘要】：目的:OPRK1與OPRM1受體屬于阿片受體家族,在神經系統(tǒng)中的分布廣泛,其與AD疾病密切相關,課題前期研究提示基因甲基化與AD疾病的關聯(lián)性,本研究將繼續(xù)探索其在AD的早期階段即輕度認知障礙(MCI)中的DNA甲基化改變。此外,利用高通量芯片檢測方法識別新的疾病風險甲基化基因,為后續(xù)的研究提供新的依據。方法:在新疆地區(qū)的流行病學調查研究得到的5398例樣本中,按照年齡、性別、民族匹配的原則隨機抽取MCI和對照各96例,利用磁珠法進行基因組DNA的提取,亞硫酸氫鹽轉化后利用焦磷酸測序方法定量檢測OPRK1、OPRM1基因的甲基化水平,并且應用雙熒光素酶報告系統(tǒng)進行啟動子活性檢測。此外,來自寧波市康寧醫(yī)院6對性別年齡匹配的AD與對照樣本,利用450K芯片進行全基因的甲基化水平測定。結果:第一部分:病例對照組比較分析結果:維族和漢族中OPRK1基因甲基化與MCI沒有關聯(lián)(p0.05),性別分層結果顯示OPRK1基因高甲基化與漢族女性MCI患病相關(p=0.015);OPRM1基因,CpG1位點高甲基化與維族MCI患病相關(總體p=5.16E-05,男性p=0.015,女性p=1.20E-03),CpG2-4低甲基化與漢族MCI患病相關(總體p=0.002,男性p=0.018,女性p=4.98E-02)。不同民族間的甲基化比較結果:兩個民族中基本資料存在差異,應用logistic回歸對其進行校正,OPRK1甲基化在MCI與對照族中均沒有民族差異(p0.05),OPRM1基因中CpG1位點甲基化在病例組中存在民族差異(CpG1 p=0.002,adjust p=0.009)CpG2-4位點甲基化在對照組中存在民族差異(CpG2-4 p=0.005,adjust p=0.009)。此外,結合前期的研究,將不同地域(新疆和浙江)漢族對照人群的基因甲基化結果進行比較,OPRK1甲基化在總體及男性分組中存在地域差異,而在女性中沒有地域差異(總體p=0.006,男性p=2.31E-04,女性p0.05),OPRM1基因在新疆漢族人群中甲基化水平顯著高于浙江漢族人群中甲基化水平(CpG1總體p=2.99E-05,男性p=0.006,女性p=0.001;CpG2-4總體p=1.32E-13,男性p=1.71E-08,女性p=2.74E-05)。偏相關分析(控制組別、性別、BMI、血糖、甘油三酯、膽固醇、高密度脂蛋白膽固醇、低密度脂蛋白膽固醇因素)顯示維族中OPRM1 CpG2-4甲基化與年齡存在正相關關系(r=0.220,p=0.039)。雙熒光素酶實驗結果進一步提示,相較于陰性對照pGL3-Basic,OPRK1與OPRM1重組質粒均能促進下游螢火蟲熒光素酶的表達,OPRK1使表達水平增加2.248倍(p=0.008),OPRM1增加2.616倍(p=0.003)。第二部分:應用450K檢測6對樣本之間的甲基化水平差異,AD與對照組間甘油三酯水平差異有統(tǒng)計學意義(p=0.025),后續(xù)的差異分析中利用線性模型對其進行校正,以|delta beta|0.2且p0.05為標準,共得到41個差異甲基化位點對應于33個基因,其中18個位點高甲基化,23個位點低甲基化。結論:OPRK1高甲基化為漢族女性MCI的風險因素,并且該片段具有啟動子活性,可能存在低表達水平,環(huán)境因素對于漢族男性OPRK1甲基化存在影響,對漢族女性沒有影響。OPRM1特定位點甲基化與不同民族的MCI風險相關,CpG1位點高甲基化是維族MCI患病的風險因素,CpG2-4位點低甲基化是漢族MCI患病的風險因素。此外,CpG1位點甲基化在MCI中存在民族差異,而CpG2-4位點甲基化在對照中存在民族差異。環(huán)境因素能夠影響OPRM1甲基化水平,表現為新疆漢族OPRM1甲基化水平高于浙江漢族;年齡是MCI重要的危險因素,維族人群中OPRM1 CpG2-4基因甲基化水平隨年齡增長而增長,OPRM1檢測片段具有啟動子活性。高通量芯片研究為后續(xù)提供新的研究依據,其中近啟動子區(qū)域的差異甲基化位點的應在擴大樣本中進行驗證,尤其應對MCI到AD的進展過程中甲基化如何改變進行研究。
[Abstract]:Objective: OPRK1 and OPRM1 receptors belong to the opioid receptor family, which are widely distributed in the nervous system and are closely related to the AD disease. Earlier studies suggest the association between gene methylation and AD disease. This study will continue to explore the changes in DNA methylation in the early stage of AD, the mild cognitive impairment (MCI). In addition, high throughput core will be used. This method identifies new disease risk methylation genes and provides a new basis for subsequent research. Methods: in 5398 samples of epidemiological studies in Xinjiang, 96 cases of MCI and 96 cases were randomly selected according to the principle of age, sex and national match, and the extraction of genomic DNA by magnetic beads and hydrogen sulfite salt After conversion, the methylation level of OPRK1, OPRM1 gene was detected by pyrosequencing method, and the promoter activity was detected by using the dual luciferase reporter system. In addition, 6 sex matched AD and control samples from Corelle hospital in Ningbo were used to determine the level of methylation of all genes by 450K chip. Results: first Part: comparison and analysis of the case control group: the OPRK1 gene methylation in the Uygur and Han nationalities was not associated with MCI (P0.05). The results of sex stratification showed that the hypermethylation of the OPRK1 gene was associated with the MCI disease of the Han women (p=0.015); the OPRM1 gene, the high methylation of the CpG1 loci was associated with the Uygur MCI disease (general p=5.16E-05, male p=0.015, female p=1.20E-03). CpG2-4 hypomethylation is associated with the prevalence of MCI in the Han nationality (overall p=0.002, male p=0.018, female p=4.98E-02). Comparison results of methylation among different ethnic groups: the basic data of the two nationalities are different, the logistic regression is used to correct them. There is no national difference (P0.05) in OPRK1 methylation in MCI and the control group, and CpG1 loci in the OPRM1 gene. Methylation in the case group has ethnic differences (CpG1 p=0.002, adjust p=0.009) CpG2-4 locus methylation (CpG2-4 p=0.005, adjust p=0.009) in the control group (CpG2-4 p=0.005, adjust p=0.009). In addition, the results of the gene methylation of the Han control group in different regions (Xinjiang and Zhejiang) were compared with the previous studies, and OPRK1 methylation was in general and in general. There were regional differences in the male group, while there was no regional difference among women (total p=0.006, male p=2.31E-04, female P0.05). The methylation level of OPRM1 gene in Xinjiang Han population was significantly higher than that in the Han population of Zhejiang (CpG1 overall p=2.99E-05, male P =0.006, female p=0.001; CpG2-4 p=1.32E-13, male p=1.71E-08). P=2.74E-05. Partial correlation analysis (control group, sex, BMI, blood sugar, triglyceride, cholesterol, HDL cholesterol, low density lipoprotein cholesterol) showed that there was a positive correlation between OPRM1 CpG2-4 methylation and age in the Uygur group (r=0.220, p= 0.039). The results of double luciferase experiment were further suggested, compared with negative control PGL3-Basic, OPRK1 and OPRM1 recombinant plasmids could promote the expression of luciferase in the lower reaches of the firefly. OPRK1 increased the expression level by 2.248 times (p=0.008) and OPRM1 increased by 2.616 times (p=0.003). The second part: the difference of the methylation level between the samples was detected with 450K, and the difference between AD and the level of triglyceride between the groups was statistically significant (p=0.025). In the subsequent difference analysis, a linear model was used to correct it. With |delta beta|0.2 and P0.05 as the standard, 41 different methylation sites were matched to 33 genes, of which 18 loci were methylation and 23 loci were low methylation. Conclusion: OPRK1 hypermethylation is the risk factor for the female MCI of the Han nationality, and the fragment has the promoter activity. Sex, may have low level of expression, environmental factors have influence on OPRK1 methylation in Han men, and there is no effect on the risk of MCI risk in the.OPRM1 specific location methylation of Han women. High methylation at the CpG1 locus is a risk factor for the MCI disease of the Uygur nationality, and the low methylation of CpG2-4 loci is the risk factor of the Han nationality in the Han nationality. In addition, C There are ethnic differences in the methylation of pG1 loci in MCI, and there are ethnic differences in the methylation of CpG2-4 loci. Environmental factors can affect the level of OPRM1 methylation, which shows that the level of OPRM1 methylation in Xinjiang Han is higher than that of the Han people in Zhejiang; age is an important risk factor for MCI, and the methylation level of OPRM1 CpG2-4 gene in the Uygur population varies with age. The OPRM1 detection fragment has the promoter activity. High throughput chip research provides a new research basis for follow-up, in which the differential methylation sites in the near promoter region should be verified in the expanded sample, especially in response to the change of methylation in the progress of MCI to AD.
【學位授予單位】：寧波大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R749.1

【相似文獻】

相關期刊論文 前10條

1 張曉云;蔣偉文;;全基因組甲基化研究進展[J];臨床口腔醫(yī)學雜志;2013年10期

2 陳亞軍;文格波;;蛋白質精氨酸甲基轉移酶的研究進展[J];國際病理科學與臨床雜志;2008年06期

3 安健;明樹紅;;抑癌基因甲基化與肺癌的診斷和治療[J];醫(yī)學綜述;2008年21期

4 張亮;肺癌與抑癌基因甲基化關系的研究進展[J];臨床與實驗病理學雜志;2003年02期

5 張志勇;李春宏;仇小強;農清清;何敏;覃健;蔣貴發(fā);黃明立;;廣西長壽地區(qū)人群白細胞DNA總體甲基化水平研究[J];中國老年學雜志;2009年12期

6 景志亮;趙穎海;;抑癌基因甲基化與肺癌[J];西南軍醫(yī);2009年05期

7 陸璐;張?zhí)m;王丹慧;蔡彥寧;;小鼠α-synuclein基因內含子1甲基化定量檢測方法的建立及不同腦區(qū)甲基化水平的比較研究[J];中國比較醫(yī)學雜志;2014年04期

8 徐酩;呂京o，

本文編號：2143486