不同条件处理对空肠弯曲菌粘附侵染的影响

发布时间：2018-05-17 01:15

本文选题：空肠弯曲杆菌 + 结肠癌上皮细胞HCT-8　；参考：《天津科技大学》2017年硕士论文

【摘要】：空肠弯曲菌是一种食源性病菌,广泛存在于鸟类、家禽的肠道内以及自然环境中,通过入侵肠上皮细胞,引发肠道炎症,还可能引起继发性的脑膜炎、关节败血症、格林-巴利综合症等。大量数据表明,空肠弯曲菌对人的感染率不低于志贺氏菌和沙门氏菌,严重威胁着食品安全和人类的健康,但目前对空肠弯曲菌的致病机制及毒力因子还不明确。本课题旨在通过研究不同培养条件对空肠弯曲菌生长以及侵染方面的影响,在前人的研究基础上进一步地去了解空肠弯曲菌的致病机理,阐明空肠弯曲菌的致病特点和生存特性,为该菌的防控提供一定的依据。首先比较空肠弯曲菌在NaCl、KCl、MgCl_2、MnCl_2、CaCl_2、胆盐、胆酸及不同pH条件下的生长情况,结果发现加入胆盐、胆酸对空肠弯曲菌的生长几乎没有影响,加入1%的NaCl、KCl、MgCl_2和pH为6条件下培养的空肠弯曲菌会延长空肠弯曲菌到稳定期的时间。其次,用1%的NaCl、KCl、MgCl_2和用乳酸、盐酸调节的pH为6条件下培养的空肠弯曲菌ATCC33560侵染结肠癌上皮细胞HCT-8,结果表明用盐酸调节的pH为6条件下培养的空肠弯曲菌侵染结肠癌上皮细胞后的侵染率最高。构建了空肠弯曲菌ATCC33560体外侵染结肠癌上皮细胞的模型。选取一般条件下培养的空肠弯曲菌(对照组),pH为7.0条件下粘附和侵染状态下的菌体(粘附组)及pH为6.0条件下培养并完成粘附和侵染的菌体(酸耐受组),利用第二代高通量测序技术测定该菌在以上状态的转录谱。实验结果表明在黏附侵染状态下,差异表达的基因主要包括与鞭毛合成相关的基因、脂多糖合成相关基因、铁获取相关基因、脂肪酸代谢相关基因、色氨酸代谢相关基因以及一些假定蛋白的基因上调。与糖代谢、TCA循环相关的基因下调。酸耐受虽然提高了该菌的侵染率,但与一般条件培养的黏附侵染状态相比,差异基因不多。选取部分基因的转录组数据,使用实时定量PCR进行验证,结果显示转录组测序数据真实可信。本研究提供的空肠弯曲杆菌在酸性条件下侵染结肠癌上皮细胞模型中的基因表达图谱信息,将有助于阐明其在胃肠感染过程中的致病机制。
[Abstract]:Campylobacter jejuni is a foodborne bacterium widely found in the intestines of birds and poultry and in the natural environment. By invading intestinal epithelial cells, Campylobacter jejuni causes intestinal inflammation, and may also lead to secondary meningitis and septicemia. Guillain-Barre syndrome, etc. A large number of data show that the infection rate of Campylobacter jejuni to human is not lower than that of Shigella and Salmonella, which is a serious threat to food safety and human health. However, the pathogenic mechanism and virulence factors of Campylobacter jejuni are not clear. The purpose of this study was to study the effects of different culture conditions on the growth and infection of Campylobacter jejuni, and to further understand the pathogenic mechanism of Campylobacter jejuni on the basis of previous studies. To elucidate the pathogenic and survival characteristics of Campylobacter jejuni and provide some basis for the prevention and control of Campylobacter jejuni. First, the growth of Campylobacter jejuni in NaCl-KCl-MgCl-2MnCl2CaCl2, bile salt, cholic acid and different pH conditions were compared. The results showed that cholic acid had little effect on the growth of Campylobacter jejuni. The Campylobacter jejuni cultured with 1% NaCl-KCl-MgCl2 and pH 6 could prolong the time from Campylobacter jejuni to stable stage. Second, 1% NaCl-KCl-MgCl2 and lactic acid, HCT-8 was infected by Campylobacter jejuni ATCC33560 cultured at pH 6. The results showed that the infection rate of Campylobacter jejuni infected colon cancer epithelial cells was the highest. A model of campylobacter jejuni ATCC33560 infecting colon cancer epithelial cells in vitro was established. The adherence and infection of Campylobacter jejuni cultured under general conditions (control group pH 7.0) and pH 6.0 (acid-tolerant group) were selected. The second generation high-throughput sequencing technique was used to determine the transcriptional profile of the bacteria in the above states. The results showed that the differentially expressed genes included those related to flagellum synthesis, lipopolysaccharide biosynthesis, iron acquisition and fatty acid metabolism. Tryptophan metabolism-related genes and some hypothetical proteins are up-regulated. Genes associated with TCA cycle of glucose metabolism are down-regulated. Although acid tolerance increased the infection rate of the strain, there were not many differentially expressed genes in comparison with the adherent infection status in general culture. The transcriptome data of some genes were selected and verified by real-time quantitative PCR. The results showed that the transcriptome sequencing data were true and reliable. The information of gene expression map of Campylobacter jejuni infected colon cancer epithelial cell model in acidic condition will be helpful to elucidate the pathogenicity of Campylobacter jejuni in the process of gastrointestinal infection.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378

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