本文選題：大腸桿菌　切入點：腸毒素　出處：《上海交通大學》2011年博士論文　論文類型：學位論文

【摘要】：產(chǎn)腸毒素大腸桿菌(enterotoxigenic Escherichia coli, ETEC)是引起動物和人體發(fā)生腹瀉的重要病原菌之一。這種病原菌分泌在表面的黏附素可以介導細菌向腸道黏膜附著,隨后細菌產(chǎn)生腸毒素而引起宿主腹瀉。乳酸菌作為一類益生菌能夠幫助宿主改善腸道菌群微生態(tài)平衡,維護宿主的腸道健康。已有的體外研究報道ETEC可以引起某些腸上皮細胞的死亡,并且乳酸菌能夠通過和ETEC的相互作用而保護宿主細胞。因此,研究ETEC對腸上皮細胞致病的分子機制,以及乳酸菌保護宿主細胞的機理,對于益生菌在防治產(chǎn)腸毒素大腸桿菌引起的腹瀉疾病方面有著重要的作用。 本研究將一株豬小腸上皮細胞系IPEC-J2細胞和兩種濃度的產(chǎn)腸毒素大腸桿菌ETEC K88菌株JG280共培養(yǎng)后,發(fā)現(xiàn)108 CFU/ml的ETEC對IPEC-J2細胞的細胞毒性顯著性高于109 CFU/ml,這一結(jié)果提示ETEC對IPEC-J2的細胞毒性根據(jù)細菌濃度的不同而產(chǎn)生差別,并且細菌的群體感應(quorum sensing)可能在ETEC的致病機理中發(fā)揮重要的作用。研究發(fā)現(xiàn)在108 CFU/ml的ETEC和IPEC-J2細胞共培養(yǎng)過程中,ETEC產(chǎn)生的AI-2(autoinducer-2,自體誘導物)活性和IPEC-J2的細胞死亡呈正相關(guān),而與ETEC的毒力基因estA(編碼大腸桿菌耐熱腸毒素a)和estB(編碼大腸桿菌耐熱腸毒素b)表達呈負相關(guān)。為了進一步地研究ETEC群體感應的機理,我們將ETEC K88菌株JG280的luxS基因(該基因的產(chǎn)物催化AI-2的生物合成)克隆,并在大腸桿菌E. coli DH5α中過量表達。將載有過量表達的luxS基因的E. coli DH5α的無菌培養(yǎng)上清液(內(nèi)含高活性的AI-2)和IPEC-J2細胞及108 CFU/ml的ETEC共培養(yǎng)后發(fā)現(xiàn),AI-2可以顯著性地降低ETEC對IPEC-J2的細胞毒性并抑制estA基因的表達。以上結(jié)果共同提示,由AI-2介導的群體感應在ETEC的致病機理中起重要作用,并且AI-2可能是通過對大腸桿菌耐熱腸毒素a的負向調(diào)控來實現(xiàn)的。 我們還研究了13株從豬體內(nèi)分離到的乳酸菌是否能夠保護IPEC-J2細胞免受ETEC K88菌株JG280的侵染,及其作用的分子機制。本研究首先發(fā)現(xiàn)一株非產(chǎn)腸毒素大腸桿菌E. coli K88 JFF4在濃度為108和109 CFU/ml時,均不會對IPEC-J2產(chǎn)生細胞毒性,這一結(jié)果提示腸毒素對于引起腸上皮細胞死亡(或損傷)的重要作用。研究還發(fā)現(xiàn),這13株乳酸菌中有5株能夠顯著地降低ETEC對IPEC-J2的細胞毒性,進而保護IPEC-J2細胞。通過對6株乳酸菌CL9、CL11、CL12、K67、S33和S64的進一步研究表明,這6株乳酸菌均能夠降低ETEC誘導IPEC-J2分泌促炎因子白細胞介素-8 (interleukin-8, IL-8),并且除了K67之外的5株乳酸菌均能夠促進IPEC-J2分泌抗炎因子白細胞介素-10 (interleukin-10, IL-10)。用實時定量PCR的方法研究發(fā)現(xiàn),一株對IPEC-J2有保護作用的乳酸菌CL9能夠抑制ETEC的estA和estB基因表達。乳酸菌S8能夠顯著性地促進ETEC分泌的AI-2活性,同時它還可以降低ETEC對IPEC-J2的細胞毒性。以上結(jié)果共同提示,某些乳酸菌可能通過抑制ETEC毒力基因的表達,另一些乳酸菌可能通過分泌某些物質(zhì)影響ETEC群體感應信號分子的作用,進而降低ETEC腸毒素的產(chǎn)生,從而保護宿主細胞。同時,IPEC-J2細胞分泌的細胞因子IL-8和IL-10在乳酸菌的作用機制中起重要作用。為了進一步研究乳酸菌的作用機理,我們還需更加深入的研究。
[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.

【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R378

【引證文獻】

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本文編號：1583745