肺癌患者不同舌苔类型的舌苔微生物研究

发布时间：2018-05-13 06:22

本文选题：舌苔微生物 + 肺癌　；参考：《皖南医学院》2016年硕士论文

【摘要】：背景:舌诊是传统中医独特的诊断方法,通过辨析观察舌苔的颜色、腐腻、厚薄等不同中医舌苔类型来判断人体生理功能及病理情况。据现阶段研究结果显示,舌苔的形成机制目前尚未明确,而舌苔微生物是影响舌苔形成的重要原因。传统微生物研究的分子生物学方法如荧光定量PCR、变性梯度凝胶电泳DGGE等不仅工作量大、耗时长,且检测出的微生物丰度有限。Illumina Miseq测序技术弥补了传统分子生物技术的缺陷,对舌苔微生物丰度和多样性进行了全面分析,满足对舌苔微生物的全面认识。肺癌是我国癌症发病率和死亡率最高的肿瘤之一。目的:本课题采用Illumina Miseq测序平台对肺癌患者不同中医舌苔类型的舌苔微生物进行分析,目的为探讨肺癌患者不同中医舌苔类型和其舌苔微生物结构之间的关系及规律,进一步揭示舌苔形成原理,促进中医舌诊的客观化和标准化。方法:收集2013年12月至2014年12月2皖南医学院附属弋矶山医院肿瘤防治中心收治肺癌患者(均经病理确诊为肺癌者)41例,体检中心招募健康正常人43例。所有入选对象均签订知情同意书并获皖南医学院伦理道德委员会同意。研究对象端坐舌象仪(DS01-B)前进行舌象采集和分析后,用拭子采集舌苔并保存在EP管中留用。提取14名肺癌患者和7名健康人的舌苔样本微生物的总DNA,16S r RNA基因V3-V4高变区经PCR扩增后产物进行Illumina测序,对舌苔微生物的组成,丰度和多样性进行全面的分析。结果:1.经舌象信息采集系统分析,41名肺癌患者中常见舌苔类型有薄白苔21例,白腻苔10例,黄腻苔8例,无苔2例;43例健康人中42人为薄白苔,1人为薄黄苔。肺癌患者舌苔厚度明显高于健康人(314.86±222.47 vs.80.00±75.84,p0.01)。2.41例肺癌患者有8例厚苔和6例薄苔,43例健康人中7例,共21份样本进行后续舌苔微生物测序分析。对21份舌苔微生物样本进行Illumina测序共获得508,003个有效序列,总碱基数为224,746,932bp,平均444.26bp,其中99.99%的序列长度为401-500bp。3.稀释性曲线表明本次测序覆盖了舌苔微生物97%的菌种。多样性分析提示厚苔组舌苔微生物丰度和多样性较其他两组均低。4.门水平,厚苔组检测出12个菌门,薄苔组检测出14个菌门,对照组19个菌门,与多样性分析相符。三组中主要菌门为厚壁菌门Firmicutes、拟杆菌门Bacteriodetes、变形菌门Proteobateria、放线菌门Actinobacteria、梭杆菌Fusobacteria,占所有门类的95%以上。属水平检测厚苔组、薄苔组和健康对照组分别有132,129和123个菌属。三组中主要菌属有普氏菌Prevotella、链球菌Streptococcus、韦荣菌Veillonella、奈瑟菌Neisseria、乳酸球菌Lactococcus、流感嗜血杆菌Haemophilus、颗粒链菌Granulicatella and梭杆菌Fusobaterium。厚苔组优势菌有链球菌Streptococcus、乳酸球菌Lactococcus和放线菌Actinomyces。结论:1.舌苔微生物的组成可能与中医舌苔类型有关;2.肺癌患者舌苔微生物的多样性和差异性可能是引起患者舌苔增厚的原因之一;3.特异性的舌苔微生物检测可能成为肺癌早期诊断的特异性生物标志物。
[Abstract]:Background: tongue diagnosis is a unique diagnostic method of traditional Chinese medicine. By distinguishing and observing the color of tongue coating, greasy, thick and thin tongue coating types to judge the physiological functions and pathological conditions of the human body. According to the results of the present stage, the mechanism of the formation of tongue fur is not yet clear, and the tongue coating microorganism is the important reason for the formation of tongue coating. Tradition The molecular biology methods, such as fluorescence quantitative PCR, denaturing gradient gel electrophoresis (DGGE) and so on, are not only large and time-consuming, but also the detected microbial abundance limited.Illumina Miseq sequencing technology makes up for the defects of traditional molecular biological technology, and makes a comprehensive analysis on the abundance and diversity of the tongue coating microorganism and satisfies the tongue fur. Lung cancer is one of the most common cancer incidence and mortality in China. Objective: the purpose of this study was to analyze the tongue coating microbes of different tongue coating types of lung cancer patients by Illumina Miseq sequencing platform, in order to explore the relationship between tongue coating types of different TCM and the structure of tongue coating microorganism in lung cancer patients. To further reveal the principle of the formation of tongue coating and to promote the objectification and standardization of tongue diagnosis in traditional Chinese medicine. Methods: 41 cases of lung cancer patients (all patients with lung cancer confirmed by pathology) were collected from December 2013 to December 2014, 2 of the cancer prevention and control center of Yi La Shan Hospital Affiliated to Wangnan Medical College, and 43 healthy people were recruited in the medical center. All the selected subjects were selected. They all signed the informed consent and received the consent of the ethics and moral Committee of Wangnan Medical College. After the study of tongue image acquisition and analysis, the object of the study was collected and analyzed by the tongue image instrument (DS01-B). The tongue coating was collected with swabs and retained in the EP tube. The total DNA of the tongue fur samples of 14 lung cancer patients and 7 healthy people was extracted, and the 16S R RNA gene V3-V4 high variation area was PCR. The amplified products were sequenced by Illumina and analyzed the composition, abundance and diversity of the tongue coating microorganism. Results: 1. the common tongue coating types in 41 lung cancer patients were thin white moss, 10 white and greasy fur, 8 yellow and greasy fur, 2 cases without moss, 42 thin white moss in 43 healthy people and 1 people with thin yellow moss. The thickness of tongue coating in cancer patients was significantly higher than that of healthy people (314.86 + 222.47 vs.80.00 + 75.84, P0.01). There were 8 thick moss and 6 thin moss in.2.41 patients and 7 cases in 43 healthy people. 21 samples were sequenced in the following tongue coating microorganism. 508003 effective sequences were obtained by Illumina sequencing for 21 tongue coated microorganism samples. The total base number of the total base was the base number 224746932bp, with an average 444.26bp, 99.99% of the sequence length was 401-500bp.3. dilution curve, indicating that the sequence covered 97% of the tongue coating microorganism. The diversity analysis suggested that the abundance and diversity of the tongue fur of the thick moss group were lower than those of the other two groups, and 12 fungi were detected in the thick moss group, and 14 bacteria were detected in the thin moss group. The 19 bacteria gates in the control group were in accordance with the diversity analysis. The main bacteria in the three groups were Firmicutes, Bacteriodetes, Proteobateria, actinomycetes Actinobacteria, and Clostridium Fusobacteria, which accounted for more than 95% of all categories. The main bacteria in the three groups are Prevotella, Streptococcus Streptococcus, Wei Rongjun Veillonella, Neisseria Neisseria, Lactococcus lactis Lactococcus, Haemophilus influenzae Haemophilus, Streptococcus Fusobaterium. moss of Granulicatella and Clostridium of granular Streptococcus, Streptococcus lactis Lactococcus and actinomycetes. Actinomyces. conclusion: the composition of 1. tongue coating microbes may be related to the type of tongue coating of traditional Chinese medicine; 2. the diversity and difference of the tongue coating microorganism in the patients with lung cancer may be one of the reasons for the thickening of the tongue fur of the patients; the 3. specific tongue coating microorganism detection may be a specific biomarker for the early diagnosis of lung cancer.

【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R273

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