鼠李糖乳杆菌代谢产物预防新生大鼠大肠杆菌感染的效果和机制研究及有效成分分析

发布时间：2018-03-01 22:17

本文关键词： 新生儿败血症和脑膜炎大肠杆菌K1株肠道屏障血脑屏障益生菌代谢产物黏蛋白免疫球蛋白A　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：一、研究背景与目的败血症和脑膜炎是导致新生儿,特别是早产儿死亡的重要原因。大肠杆菌(Escherichia coli,E.coli)是引起新生儿败血症和脑膜炎最常见的革兰阴性致病菌。新生儿E.coli败血症和脑膜炎的治疗主要依靠抗生素,但疗效方面有着诸多的限制,其不合理的使用也导致耐药E.coli感染发生率逐年上升。因此,亟需积极的开发预防新生儿E.coli败血症和脑膜炎的新方法,代替以抗生素为主的治疗方法。大肠杆菌是肠道常驻菌,成功突破肠黏膜屏障是自然状态下E.coli引起败血症和脑膜炎的关键步骤。新生儿的肠道屏障功能发育不完全,极易感染E.coli继而引发败血症和脑膜炎,因此,通过外界干预增强新生儿肠道屏障功能是预防该病的有效措施。目前的研究表明,鼠李糖乳杆菌GG株(Lactobacillus rhamnosusGG,LGG)的代谢产物具有调节肠道屏障功能的效果,但能否用于预防新生儿E.coli感染仍未有研究。本研究着重探讨了 LGG培养上清液(LGG Culture Supernatant,LCS)预防新生儿E.coli感染的效果及相关机理,并对代谢产物中的有效活性成分作了初步分析。二、研究方法和结果方法:首先,培养人结肠腺癌细胞Caco-2建立体外肠屏障模型,初步研究LCS保护肠屏障功能并预防E.coli易位的效果;其次,建立新生大鼠肠源性E.coli感染模型,进一步研究LCS预防新生大鼠E.coli系统性感染的效果和相关机理;最后,通过高效液相色谱-串联质谱等技术,分析并初步鉴定LCS中发挥功效的活性成分。结果:体外研究结果显示LCS能抑制E.coli降解Caco-2分泌的黏蛋白,抑制E.coli的黏附和侵袭,减轻E.coli对肠上皮细胞间紧密连接的破坏并阻止其易位;体内研究结果显示LCS能有效预防新生大鼠肠源性E.coli感染,其机制是促进了新生大鼠肠道细胞的增殖和分化、增加了肠道表层的黏蛋白、免疫球蛋白A和细胞间紧密连接蛋白的表达并降低了肠道的透通性。这些研究结果表明LCS能促进新生大鼠肠道屏障功能的形成和完善,并藉此增强大鼠对肠源性E.coli感染的抵抗力。最后,通过高效液相色谱-串联质谱和蛋白表达与纯化技术,我们鉴定并体外表达和纯化了 LCS中的一种假想蛋白HMPREF0539_2242,后续的研究发现该蛋白很可能发挥了促进新生大鼠肠道屏障功能成熟的作用。三、结论新生儿早期肠道屏障功能的不完善主要表现为肠道机械屏障未形成、正常菌群结构未建立和肠道免疫系统不成熟,这些都为E.coli感染提供了可乘之机。在本研究中,我们首次发现LGG的代谢产物可促进新生大鼠肠道机械屏障和免疫屏障的形成,继而增强大鼠对E.coli感染的抵抗力。通过质谱分析,我们鉴定并纯化了 LCS中的一种蛋白,发现该蛋白很可能发挥了上述的益生作用。这些研究结果为开发新生儿E.coli败血症和脑膜炎的新型预防药物提供了基础。
[Abstract]:First, the background and purpose of the study was that septicemia and meningitis were the cause of newborns, E. coli Escherichia coli is the most common gram-negative pathogen causing neonatal septicemia and meningitis. The treatment of neonatal E. coli septicemia and meningitis mainly depends on antibiotics. However, there are many limitations in therapeutic effect, and its irrational use also leads to the increasing incidence of drug-resistant E. coli infection year by year. Therefore, it is urgent to actively develop new methods to prevent neonatal E. coli septicemia and meningitis. Escherichia coli is a resident enteric bacteria, and it is a key step for E. coli to successfully break through intestinal mucosal barrier in natural state to cause septicemia and meningitis. The intestinal barrier function of newborn is not fully developed. E. coli is highly susceptible to septicemia and meningitis. Therefore, enhancing the intestinal barrier function of newborns through external intervention is an effective measure to prevent the disease. The metabolites of Lactobacillus rhamnosus GGGGG have the function of regulating intestinal barrier. However, whether it can be used to prevent neonatal E. coli infection has not been studied. In this study, the effect of LGG culture supernatant, LGG Culture supernatant LCSC, on the prevention of neonatal E. coli infection and its related mechanism were discussed, and the effective active components in metabolites were preliminarily analyzed. Methods and results: firstly, the intestinal barrier model was established by cultured human colon adenocarcinoma cell line Caco-2, and the effect of LCS on protecting intestinal barrier function and preventing E. coli translocation was preliminarily studied. To further study the efficacy and related mechanism of LCS in preventing systemic infection of E. coli in newborn rats. Finally, by means of high performance liquid chromatography-tandem mass spectrometry (HPLC-MS), Results: in vitro studies showed that LCS could inhibit the degradation of mucin secreted by Caco-2 and inhibit the adhesion and invasion of E. coli. The results of in vivo study showed that LCS could effectively prevent enterogenic E. coli infection in neonatal rats, and its mechanism was to promote the proliferation and differentiation of intestinal cells in neonatal rats. It increased the expression of mucin, immunoglobulin A and intercellular tight junction protein, and decreased the permeability of intestinal tract. These results suggest that LCS can promote the formation and improvement of intestinal barrier function in neonatal rats. Finally, high performance liquid chromatography-tandem mass spectrometry (HPLC / MS) and protein expression and purification techniques were used to enhance the resistance of rats to enterogenic E. coli infection. We have identified, expressed and purified a hypothetical protein HMPREF05392242 from LCS in vitro. Subsequent studies have found that HMPREF05392242 may play a role in promoting the maturation of intestinal barrier function in neonatal rats. Conclusion the imperfections of intestinal barrier function in the early stage of newborn mainly show that the intestinal mechanical barrier is not formed, the normal flora structure is not established and the intestinal immune system is not mature, which provide the opportunity for E. coli infection. We found for the first time that the metabolites of LGG could promote the formation of intestinal mechanical and immune barrier in neonatal rats, and then enhance the resistance of rats to E. coli infection. By mass spectrometry, we identified and purified a protein in LCS. These results provide the basis for the development of new prophylaxis of neonatal E. coli septicemia and meningitis.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R722.1

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