产肠毒素大肠杆菌、肠上皮细胞和乳酸菌相互关系的研究

发布时间：2018-03-08 11:39

本文选题：大肠杆菌　切入点：肠毒素　出处：《上海交通大学》2011年博士论文　论文类型：学位论文

【摘要】：产肠毒素大肠杆菌(enterotoxigenic Escherichia coli, ETEC)是引起动物和人体发生腹泻的重要病原菌之一。这种病原菌分泌在表面的黏附素可以介导细菌向肠道黏膜附着,随后细菌产生肠毒素而引起宿主腹泻。乳酸菌作为一类益生菌能够帮助宿主改善肠道菌群微生态平衡,维护宿主的肠道健康。已有的体外研究报道ETEC可以引起某些肠上皮细胞的死亡,并且乳酸菌能够通过和ETEC的相互作用而保护宿主细胞。因此,研究ETEC对肠上皮细胞致病的分子机制,以及乳酸菌保护宿主细胞的机理,对于益生菌在防治产肠毒素大肠杆菌引起的腹泻疾病方面有着重要的作用。本研究将一株猪小肠上皮细胞系IPEC-J2细胞和两种浓度的产肠毒素大肠杆菌ETEC K88菌株JG280共培养后,发现108 CFU/ml的ETEC对IPEC-J2细胞的细胞毒性显著性高于109 CFU/ml,这一结果提示ETEC对IPEC-J2的细胞毒性根据细菌浓度的不同而产生差别,并且细菌的群体感应(quorum sensing)可能在ETEC的致病机理中发挥重要的作用。研究发现在108 CFU/ml的ETEC和IPEC-J2细胞共培养过程中,ETEC产生的AI-2(autoinducer-2,自体诱导物)活性和IPEC-J2的细胞死亡呈正相关,而与ETEC的毒力基因estA(编码大肠杆菌耐热肠毒素a)和estB(编码大肠杆菌耐热肠毒素b)表达呈负相关。为了进一步地研究ETEC群体感应的机理,我们将ETEC K88菌株JG280的luxS基因(该基因的产物催化AI-2的生物合成)克隆,并在大肠杆菌E. coli DH5α中过量表达。将载有过量表达的luxS基因的E. coli DH5α的无菌培养上清液(内含高活性的AI-2)和IPEC-J2细胞及108 CFU/ml的ETEC共培养后发现,AI-2可以显著性地降低ETEC对IPEC-J2的细胞毒性并抑制estA基因的表达。以上结果共同提示,由AI-2介导的群体感应在ETEC的致病机理中起重要作用,并且AI-2可能是通过对大肠杆菌耐热肠毒素a的负向调控来实现的。我们还研究了13株从猪体内分离到的乳酸菌是否能够保护IPEC-J2细胞免受ETEC K88菌株JG280的侵染,及其作用的分子机制。本研究首先发现一株非产肠毒素大肠杆菌E. coli K88 JFF4在浓度为108和109 CFU/ml时,均不会对IPEC-J2产生细胞毒性,这一结果提示肠毒素对于引起肠上皮细胞死亡(或损伤)的重要作用。研究还发现,这13株乳酸菌中有5株能够显著地降低ETEC对IPEC-J2的细胞毒性,进而保护IPEC-J2细胞。通过对6株乳酸菌CL9、CL11、CL12、K67、S33和S64的进一步研究表明,这6株乳酸菌均能够降低ETEC诱导IPEC-J2分泌促炎因子白细胞介素-8 (interleukin-8, IL-8),并且除了K67之外的5株乳酸菌均能够促进IPEC-J2分泌抗炎因子白细胞介素-10 (interleukin-10, IL-10)。用实时定量PCR的方法研究发现,一株对IPEC-J2有保护作用的乳酸菌CL9能够抑制ETEC的estA和estB基因表达。乳酸菌S8能够显著性地促进ETEC分泌的AI-2活性,同时它还可以降低ETEC对IPEC-J2的细胞毒性。以上结果共同提示,某些乳酸菌可能通过抑制ETEC毒力基因的表达,另一些乳酸菌可能通过分泌某些物质影响ETEC群体感应信号分子的作用,进而降低ETEC肠毒素的产生,从而保护宿主细胞。同时,IPEC-J2细胞分泌的细胞因子IL-8和IL-10在乳酸菌的作用机制中起重要作用。为了进一步研究乳酸菌的作用机理,我们还需更加深入的研究。
[Abstract]:Enterotoxigenic Escherichia coli (enterotoxigenic Escherichia, coli, ETEC) is one of the important pathogens of human and animal pathogens of diarrhea. The secretion on the surface adhesion mediated by bacterial attachment to the intestinal mucosa, then the bacteria to produce enterotoxin caused by host diarrhea. As a kind of probiotic lactic acid bacteria can help improve intestinal bacteria host group of micro ecological balance, maintaining the host intestinal health. The in vitro studies have shown that ETEC can cause intestinal epithelial cell death, and the interaction of lactic acid bacteria can be through ETEC and while protecting the host cell. Therefore, the molecular mechanism of ETEC on intestinal epithelial cells in the disease, and the protective mechanism of lactic acid bacteria in host cells. For probiotics play an important role in disease prevention and treatment of diarrhea caused by enterotoxigenic Escherichia coli.
This study will be a strain of swine intestinal epithelial cell line IPEC-J2 cells and two concentrations of enterotoxigenic Escherichia coli ETEC were co cultured with JG280 K88 strain, found 108 cytotoxicity of CFU/ml ETEC of IPEC-J2 were significantly higher than that of 109 CFU/ml, these results suggest that the cytotoxicity of ETEC to IPEC-J2 and the difference according to different bacterial concentrations, quorum sensing bacteria (quorum and sensing) may play an important role in the pathogenesis of ETEC. The study found that 108 CFU/ml in ETEC and IPEC-J2 cells were co cultured in ETEC produced by AI-2 (autoinducer-2, autoinducer) activity and IPEC-J2 cell death was positively correlated with the virulence gene estA ETEC (encoding Escherichia coli enterotoxin (a) and estB encoding Escherichia coli heat stable enterotoxin B) expression was negatively correlated. In order to further study the mechanism of ETEC induction group, we will ETEC K88 The luxS gene of strain JG280 (biosynthetic product of this gene by AI-2) clone, and overexpression in Escherichia coli E. alpha DH5 coli. The luxS gene containing sterile overexpression of E. coli DH5 alpha supernatant (containing high activity AI-2) co cultured with IPEC-J2 cells and 108 CFU/ml after ETEC found AI-2, can significantly reduce the cytotoxicity of ETEC to IPEC-J2 and inhibit the expression of estA gene. The results showed that quorum sensing mediated by AI-2 play an important role in the pathogenesis of ETEC, and AI-2 may be based on the Escherichia coli enterotoxin negative a to realize to control.
We also studied whether the infection of lactic acid bacteria isolated from pigs can protect IPEC-J2 cells from ETEC K88 strain JG280 13 strains, molecular mechanism and effect. This study first found a non enterotoxigenic Escherichia coli E. coli K88 JFF4 at the concentration of 108 and 109 CFU/ml, were not cytotoxic to IPEC-J2 these results suggest that, for intestinal epithelial cell death caused by enterotoxin (or damage) of the important role. The study also found that these 13 strains of lactic acid bacteria in 5 strains could significantly reduce the cytotoxicity of ETEC to IPEC-J2, in order to protect IPEC-J2 cells. The 6 strains of lactic acid bacteria CL9, CL11, CL12, K67. The further study of S33 and S64 showed that the 6 strains of lactic acid bacteria can reduce ETEC induced IPEC-J2 secretion of proinflammatory cytokine interleukin -8 (interleukin-8, IL-8), and in addition to the 5 strains of lactic acid bacteria K67 could promote the secretion of anti-inflammatory IPEC-J2 Factor interleukin -10 (interleukin-10, IL-10). The study found that using the method of real-time quantitative PCR, the expression of estA and estB gene of lactic acid bacteria CL9 strain has a protective effect on IPEC-J2 can inhibit ETEC. Lactic acid bacteria S8 can significantly promote ETEC secretion activity of AI-2, and it can also decrease the cytotoxicity of ETEC for IPEC-J2. The above results showed that some lactic acid bacteria probably by inhibiting ETEC expression of virulence genes, some lactic acid bacteria can secrete some substances affecting ETEC quorum sensing signal molecules, thereby reducing the ETEC enterotoxin production, thereby protecting the host cell. At the same time, IPEC-J2 cells secrete cytokines IL-8 and IL-10 play important role in the mechanism of lactic acid bacteria. In order to further studying the mechanism of lactic acid bacteria, we also need further study.

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R378

【引证文献】