支气管哮喘唾液、诱导痰与粪便的细菌群谱研究

发布时间：2018-04-19 17:01

本文选题：哮喘 + 唾液　；参考：《广州医科大学》2017年硕士论文

【摘要】：第一部分支气管哮喘唾液、诱导痰及粪便细菌群谱多样性及相似性分析背景:支气管哮喘是多种炎症细胞参与的气道炎症性疾病,其疾病进程与遗传、环境、机体的免疫状态等因素具有密切的联系,而微生物在其中扮演了重要的作用。随着16s RNA基因测序技术的发展,人们发现健康人口腔、呼吸道及消化道之间的细菌群谱多样性存在差异,而同时它们亦具有不同程度的相似性,表明三者之间细菌存在相互影响。哮喘人群口腔、呼吸道及消化道细菌群谱结构的差异及相似性研究较为缺乏,其与哮喘的关系尚不明确。目的:比较哮喘唾液、诱导痰及粪便之间细菌群谱多样性及相似性。方法:随机入组55例哮喘患者,同时采集唾液、诱导痰及粪便样本进行细菌基因组DNA提取及鉴定。共32例样本经鉴定合格并进行细菌16S r RNA基因V4区的扩增及文库构建。在Miseq250平台(BGI,武汉)对扩增样品进行可逆终止链合成测序。测序所得数据与物种数据库进行比对,行细菌OTU的物种注释,分析唾液、诱导痰及粪便之间细菌群谱多样性及相似性。结果:1.在细菌基本测序数据及OTU统计分析方面,粪便原始读取数及有效读取数均显著高于诱导痰(p0.05),诱导痰有效读取率同时高于粪便及唾液,而唾液有效读取率则显著低于粪便(均p0.05)。粪便OTU总数显著高于诱导痰及唾液(p0.05)。2.在Alpha多样性分析方面,哮喘唾液细菌Shannon指数高于粪便,而诱导痰细菌Simspon指数高于粪便(均p0.05)。3.在Beta多样性分析方面,粪便与唾液细菌群谱多样性差异最大,其次为粪便与诱导痰,唾液与诱导痰细菌群谱多样性差异最小(R2=0.5081,p0.001)。4.唾液与粪便细菌相似性指数最高,其次为诱导痰与粪便,唾液与诱导痰细菌相似性指数最低。其中唾液-粪便及诱导痰-粪便、唾液-粪便及唾液-诱导痰之间相似性指数具有显著性差异(均p0.05)。结论:哮喘唾液、诱导痰及粪便细菌群谱多样性存在差异。同时,唾液、诱导痰及粪便细菌存在不同程度相似性。其中唾液与粪便细菌群落结构及相对丰度综合相似性最高。第二部分诱导痰及粪便细菌群谱与支气管哮喘临床特征的相关性分析背景:人类呼吸道过去被认为处于无菌环境中,而微生物基因检测技术的应用使人们认识到呼吸道内存在大量细菌定植。而大量研究表明,健康人与哮喘患者呼吸道细菌群谱多样性存在差异且其与哮喘临床特征存在明显相关性。而“卫生假说”则提出生命早期消化道细菌群谱多样性的改变可导致成年哮喘发病风险增高。因此呼吸道及消化道细菌群谱的变化与哮喘疾病进程具有密切关系。目前该领域研究多集中与国外哮喘人群,中国人群呼吸道及消化道细菌群谱与哮喘的关系尚不明确。目的:比较哮喘与健康人诱导痰及粪便的细菌群谱的差异及其与哮喘临床特征的相关性。方法:诱导痰检测:随机入选41例哮喘及22例健康人进行细菌基因检测及鉴定。粪便检测:随机入选38例哮喘及24例健康人进行细菌基因检测及鉴定。所有受试者行肺功能、诱导痰、血常规及专项过敏原检测。经鉴定分别有23例哮喘与14例健康人诱导痰样本、29例哮喘与15例健康人粪便样本检测合格并进行细菌16S r RNA基因V4区的扩增及文库构建。在Miseq250平台(BGI,武汉)对扩增样品进行可逆终止链合成测序。测序所得数据与物种数据库进行比对,行细菌OTU的物种注释,分析哮喘诱导痰及粪便细菌群谱多样性与健康人的差异,同时将其与临床指标进行相关性分析。结果:1.诱导痰:(1)临床特征:与健康对照组相比,哮喘组年龄[50.0(40.6,52.3)]、痰嗜酸性粒细胞比率[9.3(11.9,31.7)]、血嗜酸粒细胞个数[0.3(0.2,0.5)]、血嗜酸粒细胞比率[4.6(3.7,7.0)]及总Ig E[172.0(124.8,,486.9)]均明显增高。而FEV1占预计值%(77.4±3.9 vs 94.1)、FEV1/FVC%(68.0±2.3)、FEF25-75占预计值%[36.9(31.6,46.4)]、MEF75占预计值%[50.9(41.5,66.6)]、MEF50占预计值%(39.2±3.8vs 77.1)及MEF25占预计值%[26.4(43.3)]则明显降低。(2)哮喘组及健康对照组诱导痰细菌Alpha多样性指数差异均无统计学意义。(3)在细菌门、纲、目水平上,哮喘组诱导痰梭杆菌丰度均明显低于健康对照组[7.360(5.888,9.758)vs11.437(7.578,21.956),p=0.042]。(4)诱导痰细菌群谱与哮喘临床特征无明显相关性。2.粪便:(1)临床特征:与健康对照组相比,哮喘组年龄(47.7±2.5岁vs 36.1)、诱导痰嗜酸粒细胞比率[12.4(13.2,29.0)]、血嗜酸粒细胞个数[0.3(0.2,0.4)]、血嗜酸粒细胞比率[4.4(3.2,6.5)]及总Ig E[158(89.1,549.9)]均显著增高,而诱导痰巨噬细胞比率[16.6(16.5,34.3)]、FEV1占预计值%(72±3.8vs95)、FEV1/FVC(66.1±2.1)、FEF25-75占预计值%[35.8(28.9,44.6)]、MEF75占预计值%[50.8(40.2,64.2)]、MEF50占预计值%[39(31.1,47.9)]及MEF25占预计值%(34.4±3.3vs77.6)均明显降低。(2)哮喘组及健康对照组粪便细菌Alpha多样性指数差异均无统计学意义。(3)在细菌门水平上,哮喘组粪便柔壁菌丰度明显低于健康对照组[0.004(-0.082,0.709)vs0(-0.003,0.130),p=0.033]。在细菌科水平上,哮喘组粪便毛螺菌丰度显著低于健康对照组(8.883±0.916vs12.005±1.155,p=0.04),而氨基酸球菌丰度则明显高于健康对照组[3.623(1.849,6.872)vs1.382(0.899,4.093),p=0.03]。(4)哮喘粪便中氨基酸球菌科丰度与FEV1(R=0.391,p=0.036)及MEF75%(R=0.658,P=0.001)呈正相关;毛螺菌科丰度与痰嗜酸粒细胞比率(R=-0.420,p=0.019)、血嗜酸粒细胞数(R=-0.465,p=0.013)及吸入性糖皮质激素剂量(R=-0.417,p=0.024)呈负相关。结论:与健康对照组相比,支气管哮喘存在不同的呼吸道及消化道细菌群谱组成,其中消化道细菌群谱与哮喘临床特征相关,而呼吸道细菌群谱与哮喘临床特征则未发现明显相关性。
[Abstract]:The analysis of the diversity and similarity of bacterial group spectrum in the first part of bronchial asthma, induced sputum and feces: bronchial asthma is an airway inflammatory disease involved in many inflammatory cells, and its disease process is closely linked with heredity, environment, and the immune state of the body, and microbes play an important role in it. With the development of 16S RNA gene sequencing technology, the diversity of bacterial group diversity between the oral, respiratory and digestive tract of healthy people is found to be different, and they also have different degrees of similarity, indicating the interaction between the three bacteria. The differences and similarities of the bacterial group structure of the oral, respiratory and digestive tract of the asthmatic population The relationship between sex study and asthma is not clear. Objective: To compare the diversity and similarity of bacterial group spectrum between sputum and feces in asthma. Methods: 55 patients with asthma were randomly selected to collect saliva, induced sputum and fecal samples for genomic DNA extraction and identification. A total of 32 samples were identified and carried out. The amplification and library construction of the bacterial 16S R RNA gene V4 region. The amplified samples were sequenced on the Miseq250 platform (BGI, Wuhan). The data were compared with the species database, the species annotation of the bacterial OTU, the analysis of saliva, the diversity and similarity of the bacterial group spectrum between the induced sputum and the feces. Results: 1. in the bacterial basis The original reading number and effective reading number of the excrement were significantly higher than that of the induced sputum (P0.05). The effective reading rate of the induced sputum was higher than that of the feces and saliva, while the effective reading rate of the saliva was significantly lower than that of the feces (both P0.05). The total number of fecal OTU was significantly higher than that of the induced sputum and saliva (P0.05).2. in Alpha diversity analysis. The Shannon index of saliva bacteria in asthma was higher than that in feces, while the Simspon index of induced sputum bacteria was higher than that of feces (P0.05).3. in Beta diversity analysis, the diversity of bacterial diversity in feces and saliva was the largest, followed by fecal and induced sputum, and the diversity of bacterial diversity in saliva and induced sputum was the smallest (R2=0.5081, p0.001).4. saliva and feces The similarity index of bacteria was the highest, followed by induced sputum and feces, and the similarity index between saliva and induced sputum bacteria was the lowest. The similarity index between saliva feces and induced sputum, saliva feces and saliva induced sputum was significantly different (all P0.05). Conclusion: the diversity of asthma saliva, induced sputum and fecal bacteria was poor. At the same time, saliva, induced sputum and fecal bacteria have different degrees of similarity. Among them, the composition and relative abundance of the bacteria community structure and relative abundance of saliva and feces are the highest. Second the correlation analysis of the correlation between the bacterial group spectrum of the induced sputum and the feces and the clinical characteristics of bronchial asthma: the human respiratory tract was considered to be in a sterile environment in the past. The application of microbiological gene detection technology has made people realize that there are a large number of bacterial colonization in the respiratory tract. A large number of studies have shown that there is a significant difference in the diversity of the bacterial group spectrum in the respiratory tract between healthy and asthmatic patients and it has a significant correlation with the clinical characteristics of asthma. The changes in the respiratory and digestive tract bacterial group spectrum are closely related to the process of asthma. At present, the research in this field is mostly focused on the people with asthma in foreign countries. The respiratory and digestive tract bacterial group spectrum of the Chinese population and the relationship between asthma are not clear. Objective: to compare the asthma and healthy people. The difference of the bacterial group spectrum of induced sputum and feces and the correlation with the clinical characteristics of asthma. Methods: induced sputum detection: 41 cases of asthma and 22 healthy people were randomly selected for bacterial gene detection and identification. Fecal detection: 38 cases of asthma and 24 healthy people were randomly selected for the detection and identification of bacterial basis. All the subjects were treated with lung function. The sputum, blood routine and specific allergen were detected in 23 cases of asthma and 14 healthy people, 29 cases of asthma and 15 healthy human feces samples were qualified and the amplification and library construction of the 16S R RNA gene V4 region were carried out. The sequence of reversible terminated chain sequence of the amplified samples was sequenced in Miseq250 platform (BGI, Wuhan). The data were compared with the species database, and the species annotation of bacterial OTU was used to analyze the diversity of the asthma induced phlegm and fecal bacterial diversity and the healthy people. At the same time, the correlation analysis was carried out with the clinical indicators. Results: 1. induced sputum: (1) clinical characteristics: compared with the healthy control group, the asthma group was [50.0 (40.6,52.3)], phlegm eosinophilia. Granulocyte ratio [9.3 (11.9,31.7)], blood eosinophil number [0.3 (0.2,0.5)], blood eosinophil ratio [4.6 (3.7,7.0) and total Ig E[172.0 (124.8, 486.9)] were significantly increased, while FEV1 accounted for estimated% (77.4 + 3.9 vs 94.1), FEV1/FVC% (68 + 2.3), FEF25-75 accounted for expected value]. The estimated value% (39.2 + 3.8vs 77.1) and MEF25 accounted for%[26.4 (43.3)] decreased significantly. (2) there was no significant difference in the Alpha diversity index of the induced sputum bacteria in the asthma group and the healthy control group. (3) the abundance of Clostridium sputum induced by the asthma group was significantly lower than that of the healthy control group [7.360 (5.888,9.758) vs11.437 (7.) (7.). 578,21.956), p=0.042]. (4) induced phlegm bacterial group spectrum has no significant correlation with clinical characteristics of asthma.2. feces: (1) clinical characteristics: compared with the healthy control group, the age of the asthma group (47.7 + 2.5 year old vs 36.1), the induced phlegm eosinophil ratio [12.4 (13.2,29.0)], the blood eosinophil number [0.3 (0.2,0.4)], the blood eosinophil ratio [4.4 (3.2,6.5)] And the total Ig E[158 (89.1549.9)] increased significantly, while the induced sputum macrophage ratio was [16.6 (16.5,34.3)], FEV1 accounted for the expected value% (72 + 3.8vs95), FEV1/FVC (66.1 + 2.1), FEF25-75 accounted for%[35.8 (28.9,44.6)], MEF75 accounted for the expected value. (2) there was no significant difference in the Alpha diversity index of the stool bacteria in the asthma group and the healthy control group. (3) the abundances of the faecal soft wall bacteria in the asthmatic group were significantly lower than that of the healthy control group [0.004 (-0.082,0.709) vs0 (-0.003,0.130), and the abundance of the faecal faecal spironum in the asthma group was significantly lower than that in the healthy control group. In the group (8.883 + 0.916vs12.005 + 1.155, p=0.04), the abundance of amino acid coccus was significantly higher than that of [3.623 (1.849,6.872) vs1.382 (0.899,4.093) in the healthy control group, and the abundance of amino acid cocci in p=0.03]. (4) was positively correlated with FEV1 (R=0.391, p=0.036) and MEF75% (R= 0.658), and the abundance of the family of the family spiriaceae and the ratio of phlegm eosinophils (4) 0.420, p=0.019), the blood eosinophil number (R=-0.465, p=0.013) and inhaled glucocorticoid dose (R=-0.417, p=0.024) were negatively correlated. Conclusion: compared with the healthy control group, the bronchial asthma has different respiratory and digestive tract bacterial group spectrum composition, among which the bacterial group spectrum of the acidide is related to the clinical characteristics of asthma and the respiratory tract bacterial group spectrum. There was no significant correlation with the clinical characteristics of asthma.

【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R562.25

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