【作者】 郑萍；

【导师】 白雪涛； 王强； 陈西平；

【作者基本信息】 中国疾病预防控制中心 ， 劳动卫生与环境卫生学， 2018， 博士

【摘要】 目的研究空气污染与特应性哮喘儿童和非特应性哮喘儿童表观遗传修饰和肠道微生物组特征的相关性,探索空气污染对特应性哮喘和非特应性哮喘儿童表观遗传修饰和肠道微生物组影响的标志物,为儿童哮喘的预防、治疗和干预提供依据。方法1.信息采集:本研究采用前瞻性定群研究(panel)设计,调查2016年9月至2017年11月到首都儿科研究所就诊、生活地点为北京市、5-14岁的哮喘儿童和朝阳区某小学5-14岁无哮喘疾病史的非哮喘儿童。通过问卷调查收集所有调查儿童的年龄、性别、过敏原、哮喘家族史、疾病史等信息;通过体格检查获得身高、体重信息;委托首都儿科研究所检测哮喘儿童的肺功能、呼出气一氧化氮(FeNO)等病情监测指标。2.空气污染物暴露评价:哮喘儿童的黑炭(BC)暴露数据来源于项目组的BC监测点的监测数据,监测地点为北京市朝阳区;PM10、PM2.5、NO2、O3等环境污染物的数据来源于中国环境监测总站。BC、PM10、PM2.5、NO2的暴露浓度为调查儿童样本采集日期的前3日滑动平均浓度,O3的暴露浓度为调查儿童样本采集当日的日最大8小时平均浓度。3.表观遗传修饰研究:使用质量浓度为4.5%的高渗盐水对所有调查儿童进行痰液诱导并采集痰液,利用ELLSA方法检测所有痰液样本中组蛋白H3和H4乙酰化水平,分析调查儿童组蛋白乙酰化的组间差异及空气污染对组蛋白乙酰化的影响。4.肠道微生物组16SrDNA测序:分别采集特应性哮喘儿童、非特应性哮喘儿童和非哮喘组儿童的粪便样本,基于Illumina HiSeq2500测序平台,采用双末端测序(Paired-End)策略,对样本进行16SrDNA(V4区)测序并分析调查儿童肠道微生物组的多样性、菌群结构等特征及空气污染暴露对肠道微生物组的影响。5.肠道微生物宏基因组测序:根据16SrDNA测序结果,选取部分有代表性的样本进行肠道微生物的宏基因组测序,基于Illumina HiSeq4000测序平台,利用Illumina PE150双末端测序(Paired-End)方法,对非特应性哮喘儿童、特应性哮喘儿童和非哮喘儿童的粪便样本进行宏基因组测序和功能基因注释,分析三组儿童肠道菌群基因功能及代谢通路的差异。6.统计分析:使用R(R3.4.4)软件进行统计分析:采用重复测量资料的方差分析方法对调查儿童H3、H4乙酰化水平的2次测量结果进行分析;采用多水平模型分析调查儿童的H3乙酰化水平与空气污染的相关性;采用多重线性回归模型分析调查儿童的H4乙酰化水平与空气污染的相关性;采用Wilcoxon秩和检验分析肠道微生物组的多样性差异,采用MRPP、Adnois、AMOVA方法分析肠道微生物菌群结构差异;采用Mantel test方法分析哮喘儿童肠道微生物组与组蛋白乙酰化水平和空气污染的相关性。结果1.调查儿童的组蛋白乙酰化水平:特应性哮喘儿童、非特应性哮喘儿童和非哮喘儿童痰液中组蛋白H3乙酰化水平具有显著性差异(单因素方差分析,F=13.01,P<0.01),H4乙酰化水平无显著性差异(单因素方差分析,F=0.141,P>0.01);初诊和复诊儿童的H3和H4乙酰化水平均具有显著性差异(重复测量资料的方差分析,P<0.001);BC和NO2暴露浓度与H3乙酰化水平具有显著相关性(多水平模型,P<0.001),PM10暴露浓度与H3乙酰化水平无显著相关性(多水平模型,P>0.05);BC、NO2和PM10暴露浓度与H4乙酰化水平无显著相关性(多重线性回归,P>0.05);按特应性分层分析显示:特应性是哮喘儿童组蛋白乙酰化水平的重要影响因素;BC暴露浓度与特应性哮喘儿童的H3乙酰化水平具有显著相关性(多水平模型,P=0.009)。2.儿童肠道微生物16SrDNA测序:采集调查儿童粪便样本,进行16SrDNA(V4区)测序,获得有效数据(7.64±0.77)万tags,聚类得到的OTUs数为625.2±269.0。肠道微生物的优势菌种为厚壁菌门(Firmicutes),拟杆菌门(Bacteroidetes),放线菌门(Actinobacteria),变形菌门(Proteobacteria),疣微菌门(Verrucomicrobia)。特应性哮喘儿童、非特应性哮喘儿童和非哮喘儿童肠道微生物差异具有显著性(AMOVA方法,F=3.455,P<0.001)。哮喘儿童肠道微生物的物种数量与BC暴露浓度具有边缘正相关性(Spearman相关,P=0.068),与 PM10(Spearman相关,P=0.014)和 O3(Spearman相关,P=0.025)暴露浓度具有显著负相关性。3.儿童肠道微生物宏基因组测序:粪便样本的宏基因组测序获得预测基因923,775个,其中617,878个(66.89%)基因与KEGG数据库匹配。基因功能注释结果显示:特应性哮喘和非特应性哮喘儿童在KO(KEGGOrthology)层级的功能基因的丰度有边缘显著性(Anosim方法,P=0.089);特应性哮喘儿童和非哮喘儿童肠道在KEGG pathway有44处差异,非特应性哮喘儿童与非哮喘儿童有51处差异,特应性哮喘儿童和非特应性哮喘儿童有56处差异。4.空气污染、组蛋白乙酰化水平和肠道微生物组关联性分析:采用VPA方法分析显示:环境因素对肠道微生物的影响大于遗传因素和表观遗传因素;采用Mantel test方法分析显示:门水平的肠道微生物组与BC暴露浓度有一定的相关性(r=0.048,P=0.138),与H3乙酰化水平具有显著相关性(r=0.133,P=0.048)。结论特应性哮喘儿童和非特应性哮喘儿童的表观遗传修饰及肠道微生物组显著不同;BC暴露浓度与组蛋白乙酰化和肠道微生物组均具有相关性,BC可能通过这两条途径调控哮喘的激发机制;BC暴露浓度与特应性哮喘儿童H3乙酰化水平显著相关,H3乙酰化位点有望成为特应性哮喘儿童空气污染暴露的效应标志物。更多还原

【Abstract】 ObjectiveThis study aimed to explore the association among the exposures of ambient air pollution,the characteristics of gut microbiota,and the changes in epigenetic modification in asthmatic children by panel study and check the different biomarkers of gut microbiota and epigenetic modification by strata of the atopy of asthmatics.Methods1.Data collection:From September 2016 to November 2017,physician diagnosed asthmatic children from Children’s Hospital Capital institute of Pediatrics and pupils without asthma from a primary school in Chaoyang District of Beijing were enrolled to participate in a prospective longitudinal panel study.All participants should live in Beijing for more than six months and the age of the participants should be five to fourteen years old.The status of lung function and the fractional exhaled nitric oxide(FeNO)of asthmatics were measured in the hospital at the enrollment.2.Assessment of air pollution exposures:Black Carbon(BC)exposures for the participants was estimated by the measurements of ambient air BC at the designated spots in Chaoyang District of Beijing,Information of PM10,PM2.5,NO2 and O3 were obtained from China National Environmental Monitoring Centre.3.Changes of epigenetic modification:Nebulized saline was administered to collect the induced sputum specimen of all participants by the recommended method.The acetylation levels of histone H3 and H4 of induced sputum were measured with ELLSA.Multilevel models and Multiple linear regression models were used to analyze the different effects of air pollution exposures on the level of histone acetylation in atopic asthma,non-atopic asthma,and controls.4.16SrDNA sequencing of intestinal microbiome:Faeces of participants were collected and the microbiome DNA was isolated for the analysis of 16SrDNA sequencing and microgenome sequencing of intestinal microorganism.Double terminal sequencing(Paired-End)strategy for 16SrDNA sequencing in V4 region was performed with the Illumina HiSeq2500 sequencing platform to observe the difference in the diversity and constitution of microbiota by group.5.Metagenome sequencing of intestinal microorganism:Faeces specimen were selected from those specimens for 16SrDNA sequencing for the metagenome sequencing with Illumina HiSeq4000 sequencing platform by Illumina PE150 double terminal sequencing(Paired-End).The differences in the functional genes of intestinal microflora in children among atopic asthmatics,non-atopic asthmatics,and the controls were annotated and the differences in the functional genes and metabolic pathways of intestinal microflora in the three groups were analyzed with R packages.6.Statistical analysis:R version 3.4.4 was used to ananlysis the results.T test,the one-way analysis of variance(ANOVA),multiple linear regression analysis,and stratified analysis were used to analyze the differences in measurements of the participants when they were first enrolled in the program.Tow-way ANOVA with repeated measures and multilevel regression model were used to test the changes of measurements.The differences of intestinal microorganism groups were analyzed by Adnois,LefSE methods.Mantal Testmethods was used to analysis the effects of BC,PM10,NO2 and O3 on acetylation level of histone(H3&H4)and the diversity of intestinal microflora.Results1.Measurements of histone acetylation:Two-way ANOVA test with repeated measures showed that the acetylation of histone H3 was significant different in atopic asthma,non-atopic asthma and control children(P<0.01).Mulitlevel regrression analysis showed that the differences of histone acetylation were signiciantly associated with the status of atopy and ambient air BC exposure levels(P<0.05).Stratified analysis showed that BC exposure was significantly correlated with H3 acetylation level in children with atopic asthma(P=0.009)2.16SrDNA sequencing of intestinal microbiome:V4 region of the 16SrDNA of 47 faece specimen were sequenced with the Illumina HiSeq2500 sequencing platform.The effective tags got were 76393.9 ± 7730 and the number of clusterings were 625.2±269 OTUs.The dominant bacteria of intestinal microflora were Firmicutes,Bacteroidetes,Actinobacteria,Proteobacteria,and Verrucomicrobia.AMOVA analysis was used to test the difference in the constitution of intestinal microflora.And the constitutions of microflora were significantly different by group of participants(F=3.46,P<0.001).The number of species was marginal correlated with BC exposure(P=0.068),significant correlated with PM10(P=0.014)and O3(P=0.025)exposure in children with asthma.3.Metagenome sequencing of intestinal microbiome:16 selected faece specimen were sent for metagenomic sequencing.A total of 923775 predicted genes were obtained and 617878(66.89%)genes were able to match KEGG database.The abundance of functional genes in children with atopic asthma and non-atopic asthma was marginally significant at the level of KEGG Orthology(P=0.089).As the gene annotation results,the numbers of different KEGG Pathway of intestinal microflora were 44(atopic asthma vs controls),51(non-atopic asthma vs Control)and 56(atopic asthma vs non-atopic asthma).4.Association of BC exposure,acetylation of histone,and intestinal microbiome:VPA analysis showed that environmental factors had greater influence on intestinal microbiome than genetic and epigenetic factors.Mantel test showed that air pollution(BC)exposure levels might be associated with the diversity of intestinal microbiome(r=0.048,P=0.138)and the acetylation of H3 was significantly associated with the intestinal microbiota(r=0.133,P=0.048).ConclusionEpigenetic modifications and the diversity or constitutions of intestinal microbiome were quite different between atopic and non-atopic asthmatics.BC may regulate the excitation mechanism of asthma with different phenotypes through Epigenetic modifications and intestinal microbiome.Acetylation of H3 might be one of the effect biomarkers of BC exposure for atopic asthmatics.更多还原