本文選題：空腸彎曲桿菌 + 結腸癌上皮細胞HCT-8　； 參考：《天津科技大學》2017年碩士論文

【摘要】：空腸彎曲菌是一種食源性病菌,廣泛存在于鳥類、家禽的腸道內(nèi)以及自然環(huán)境中,通過入侵腸上皮細胞,引發(fā)腸道炎癥,還可能引起繼發(fā)性的腦膜炎、關節(jié)敗血癥、格林-巴利綜合癥等。大量數(shù)據(jù)表明,空腸彎曲菌對人的感染率不低于志賀氏菌和沙門氏菌,嚴重威脅著食品安全和人類的健康,但目前對空腸彎曲菌的致病機制及毒力因子還不明確。本課題旨在通過研究不同培養(yǎng)條件對空腸彎曲菌生長以及侵染方面的影響,在前人的研究基礎上進一步地去了解空腸彎曲菌的致病機理,闡明空腸彎曲菌的致病特點和生存特性,為該菌的防控提供一定的依據(jù)。首先比較空腸彎曲菌在NaCl、KCl、MgCl_2、MnCl_2、CaCl_2、膽鹽、膽酸及不同pH條件下的生長情況,結果發(fā)現(xiàn)加入膽鹽、膽酸對空腸彎曲菌的生長幾乎沒有影響,加入1%的NaCl、KCl、MgCl_2和pH為6條件下培養(yǎng)的空腸彎曲菌會延長空腸彎曲菌到穩(wěn)定期的時間。其次,用1%的NaCl、KCl、MgCl_2和用乳酸、鹽酸調(diào)節(jié)的pH為6條件下培養(yǎng)的空腸彎曲菌ATCC33560侵染結腸癌上皮細胞HCT-8,結果表明用鹽酸調(diào)節(jié)的pH為6條件下培養(yǎng)的空腸彎曲菌侵染結腸癌上皮細胞后的侵染率最高。構建了空腸彎曲菌ATCC33560體外侵染結腸癌上皮細胞的模型。選取一般條件下培養(yǎng)的空腸彎曲菌(對照組),pH為7.0條件下粘附和侵染狀態(tài)下的菌體(粘附組)及pH為6.0條件下培養(yǎng)并完成粘附和侵染的菌體(酸耐受組),利用第二代高通量測序技術測定該菌在以上狀態(tài)的轉(zhuǎn)錄譜。實驗結果表明在黏附侵染狀態(tài)下,差異表達的基因主要包括與鞭毛合成相關的基因、脂多糖合成相關基因、鐵獲取相關基因、脂肪酸代謝相關基因、色氨酸代謝相關基因以及一些假定蛋白的基因上調(diào)。與糖代謝、TCA循環(huán)相關的基因下調(diào)。酸耐受雖然提高了該菌的侵染率,但與一般條件培養(yǎng)的黏附侵染狀態(tài)相比,差異基因不多。選取部分基因的轉(zhuǎn)錄組數(shù)據(jù),使用實時定量PCR進行驗證,結果顯示轉(zhuǎn)錄組測序數(shù)據(jù)真實可信。本研究提供的空腸彎曲桿菌在酸性條件下侵染結腸癌上皮細胞模型中的基因表達圖譜信息,將有助于闡明其在胃腸感染過程中的致病機制。
[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.
【學位授予單位】：天津科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R378

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