發(fā)布時間：2018-04-19 17:01

  本文選題：哮喘 + 唾液��； 參考：《廣州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：第一部分支氣管哮喘唾液、誘導(dǎo)痰及糞便細(xì)菌群譜多樣性及相似性分析背景:支氣管哮喘是多種炎癥細(xì)胞參與的氣道炎癥性疾病,其疾病進(jìn)程與遺傳、環(huán)境、機(jī)體的免疫狀態(tài)等因素具有密切的聯(lián)系,而微生物在其中扮演了重要的作用。隨著16s RNA基因測序技術(shù)的發(fā)展,人們發(fā)現(xiàn)健康人口腔、呼吸道及消化道之間的細(xì)菌群譜多樣性存在差異,而同時它們亦具有不同程度的相似性,表明三者之間細(xì)菌存在相互影響。哮喘人群口腔、呼吸道及消化道細(xì)菌群譜結(jié)構(gòu)的差異及相似性研究較為缺乏,其與哮喘的關(guān)系尚不明確。目的:比較哮喘唾液、誘導(dǎo)痰及糞便之間細(xì)菌群譜多樣性及相似性。方法:隨機(jī)入組55例哮喘患者,同時采集唾液、誘導(dǎo)痰及糞便樣本進(jìn)行細(xì)菌基因組DNA提取及鑒定。共32例樣本經(jīng)鑒定合格并進(jìn)行細(xì)菌16S r RNA基因V4區(qū)的擴(kuò)增及文庫構(gòu)建。在Miseq250平臺(BGI,武漢)對擴(kuò)增樣品進(jìn)行可逆終止鏈合成測序。測序所得數(shù)據(jù)與物種數(shù)據(jù)庫進(jìn)行比對,行細(xì)菌OTU的物種注釋,分析唾液、誘導(dǎo)痰及糞便之間細(xì)菌群譜多樣性及相似性。結(jié)果:1.在細(xì)菌基本測序數(shù)據(jù)及OTU統(tǒng)計(jì)分析方面,糞便原始讀取數(shù)及有效讀取數(shù)均顯著高于誘導(dǎo)痰(p0.05),誘導(dǎo)痰有效讀取率同時高于糞便及唾液,而唾液有效讀取率則顯著低于糞便(均p0.05)。糞便OTU總數(shù)顯著高于誘導(dǎo)痰及唾液(p0.05)。2.在Alpha多樣性分析方面,哮喘唾液細(xì)菌Shannon指數(shù)高于糞便,而誘導(dǎo)痰細(xì)菌Simspon指數(shù)高于糞便(均p0.05)。3.在Beta多樣性分析方面,糞便與唾液細(xì)菌群譜多樣性差異最大,其次為糞便與誘導(dǎo)痰,唾液與誘導(dǎo)痰細(xì)菌群譜多樣性差異最小(R2=0.5081,p0.001)。4.唾液與糞便細(xì)菌相似性指數(shù)最高,其次為誘導(dǎo)痰與糞便,唾液與誘導(dǎo)痰細(xì)菌相似性指數(shù)最低。其中唾液-糞便及誘導(dǎo)痰-糞便、唾液-糞便及唾液-誘導(dǎo)痰之間相似性指數(shù)具有顯著性差異(均p0.05)。結(jié)論:哮喘唾液、誘導(dǎo)痰及糞便細(xì)菌群譜多樣性存在差異。同時,唾液、誘導(dǎo)痰及糞便細(xì)菌存在不同程度相似性。其中唾液與糞便細(xì)菌群落結(jié)構(gòu)及相對豐度綜合相似性最高。第二部分誘導(dǎo)痰及糞便細(xì)菌群譜與支氣管哮喘臨床特征的相關(guān)性分析背景:人類呼吸道過去被認(rèn)為處于無菌環(huán)境中,而微生物基因檢測技術(shù)的應(yīng)用使人們認(rèn)識到呼吸道內(nèi)存在大量細(xì)菌定植。而大量研究表明,健康人與哮喘患者呼吸道細(xì)菌群譜多樣性存在差異且其與哮喘臨床特征存在明顯相關(guān)性。而“衛(wèi)生假說”則提出生命早期消化道細(xì)菌群譜多樣性的改變可導(dǎo)致成年哮喘發(fā)病風(fēng)險增高。因此呼吸道及消化道細(xì)菌群譜的變化與哮喘疾病進(jìn)程具有密切關(guān)系。目前該領(lǐng)域研究多集中與國外哮喘人群,中國人群呼吸道及消化道細(xì)菌群譜與哮喘的關(guān)系尚不明確。目的:比較哮喘與健康人誘導(dǎo)痰及糞便的細(xì)菌群譜的差異及其與哮喘臨床特征的相關(guān)性。方法:誘導(dǎo)痰檢測:隨機(jī)入選41例哮喘及22例健康人進(jìn)行細(xì)菌基因檢測及鑒定。糞便檢測:隨機(jī)入選38例哮喘及24例健康人進(jìn)行細(xì)菌基因檢測及鑒定。所有受試者行肺功能、誘導(dǎo)痰、血常規(guī)及專項(xiàng)過敏原檢測。經(jīng)鑒定分別有23例哮喘與14例健康人誘導(dǎo)痰樣本、29例哮喘與15例健康人糞便樣本檢測合格并進(jìn)行細(xì)菌16S r RNA基因V4區(qū)的擴(kuò)增及文庫構(gòu)建。在Miseq250平臺(BGI,武漢)對擴(kuò)增樣品進(jìn)行可逆終止鏈合成測序。測序所得數(shù)據(jù)與物種數(shù)據(jù)庫進(jìn)行比對,行細(xì)菌OTU的物種注釋,分析哮喘誘導(dǎo)痰及糞便細(xì)菌群譜多樣性與健康人的差異,同時將其與臨床指標(biāo)進(jìn)行相關(guān)性分析。結(jié)果:1.誘導(dǎo)痰:(1)臨床特征:與健康對照組相比,哮喘組年齡[50.0(40.6,52.3)]、痰嗜酸性粒細(xì)胞比率[9.3(11.9,31.7)]、血嗜酸粒細(xì)胞個數(shù)[0.3(0.2,0.5)]、血嗜酸粒細(xì)胞比率[4.6(3.7,7.0)]及總Ig E[172.0(124.8,,486.9)]均明顯增高。而FEV1占預(yù)計(jì)值%(77.4±3.9 vs 94.1)、FEV1/FVC%(68.0±2.3)、FEF25-75占預(yù)計(jì)值%[36.9(31.6,46.4)]、MEF75占預(yù)計(jì)值%[50.9(41.5,66.6)]、MEF50占預(yù)計(jì)值%(39.2±3.8vs 77.1)及MEF25占預(yù)計(jì)值%[26.4(43.3)]則明顯降低。(2)哮喘組及健康對照組誘導(dǎo)痰細(xì)菌Alpha多樣性指數(shù)差異均無統(tǒng)計(jì)學(xué)意義。(3)在細(xì)菌門、綱、目水平上,哮喘組誘導(dǎo)痰梭桿菌豐度均明顯低于健康對照組[7.360(5.888,9.758)vs11.437(7.578,21.956),p=0.042]。(4)誘導(dǎo)痰細(xì)菌群譜與哮喘臨床特征無明顯相關(guān)性。2.糞便:(1)臨床特征:與健康對照組相比,哮喘組年齡(47.7±2.5歲vs 36.1)、誘導(dǎo)痰嗜酸粒細(xì)胞比率[12.4(13.2,29.0)]、血嗜酸粒細(xì)胞個數(shù)[0.3(0.2,0.4)]、血嗜酸粒細(xì)胞比率[4.4(3.2,6.5)]及總Ig E[158(89.1,549.9)]均顯著增高,而誘導(dǎo)痰巨噬細(xì)胞比率[16.6(16.5,34.3)]、FEV1占預(yù)計(jì)值%(72±3.8vs95)、FEV1/FVC(66.1±2.1)、FEF25-75占預(yù)計(jì)值%[35.8(28.9,44.6)]、MEF75占預(yù)計(jì)值%[50.8(40.2,64.2)]、MEF50占預(yù)計(jì)值%[39(31.1,47.9)]及MEF25占預(yù)計(jì)值%(34.4±3.3vs77.6)均明顯降低。(2)哮喘組及健康對照組糞便細(xì)菌Alpha多樣性指數(shù)差異均無統(tǒng)計(jì)學(xué)意義。(3)在細(xì)菌門水平上,哮喘組糞便柔壁菌豐度明顯低于健康對照組[0.004(-0.082,0.709)vs0(-0.003,0.130),p=0.033]。在細(xì)菌科水平上,哮喘組糞便毛螺菌豐度顯著低于健康對照組(8.883±0.916vs12.005±1.155,p=0.04),而氨基酸球菌豐度則明顯高于健康對照組[3.623(1.849,6.872)vs1.382(0.899,4.093),p=0.03]。(4)哮喘糞便中氨基酸球菌科豐度與FEV1(R=0.391,p=0.036)及MEF75%(R=0.658,P=0.001)呈正相關(guān);毛螺菌科豐度與痰嗜酸粒細(xì)胞比率(R=-0.420,p=0.019)、血嗜酸粒細(xì)胞數(shù)(R=-0.465,p=0.013)及吸入性糖皮質(zhì)激素劑量(R=-0.417,p=0.024)呈負(fù)相關(guān)。結(jié)論:與健康對照組相比,支氣管哮喘存在不同的呼吸道及消化道細(xì)菌群譜組成,其中消化道細(xì)菌群譜與哮喘臨床特征相關(guān),而呼吸道細(xì)菌群譜與哮喘臨床特征則未發(fā)現(xiàn)明顯相關(guān)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.

【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R562.25

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本文編號：1773951