益生菌抑制大肠埃希菌K1株粘附与侵袭的机理研究

发布时间：2018-04-05 14:04

本文选题：益生菌　切入点：大肠埃希菌K1株　出处：《南方医科大学》2010年硕士论文

【摘要】： 一、研究背景和目的细菌性脑膜炎是新生儿时期中枢神经系统最常见最严重的感染。在过去三十年中,发达国家和发展中国家,细菌性脑膜炎发病率一直都没有明显变化,占新生儿疾病的1‰,但死亡率达17%-38%,未死亡者遗留神经系统后遗症者也高达58%。B族链球菌和大肠埃希菌是引起新生儿细菌性脑膜炎的最常见病原体,由于对B族链球菌携带孕妇的产时预防和对新生儿的抗生素选择性治疗,所以新生儿B族链球菌感染率明显减低,但同时也降低了新生儿对非B族链球菌感染的抵抗力,致使大肠埃希菌(E.coli)K1株成为目前新生儿细菌性脑膜炎的首要致病菌。大肠埃希菌(E. coli)K1株在40%败血症或80%脑膜炎患儿中都可分离到,它主要源于母亲肠道,孕妇因其特殊体质,肠道定殖的E. coli K1株容易易位于阴道,致使新生儿在通过产道时获得感染。研究表明新生儿20%肠道中有E. coli K1株定植,其中孕妇肠道有该菌定植的占77%。E. coli K1株一旦定植于新生儿肠道,0.5%可发生肠道侵袭性定位转移而入血,并穿过血脑屏障而引发脑膜炎。随着广谱抗生素的广泛应用,耐药性大肠埃希菌不断增多,致使新生儿尤其是低出生体重、极低出生体重儿的大肠埃希菌性脑膜炎病死率逐渐增高。抗生素耐药性已成为过去十年中一个严重的公共健康问题。补充和替代抗生素治疗的药物和方法研究也越来越受到关注。目前E. coli K1株如何通过肠屏障造成中枢神经系统感染,仍不清楚,但孕妇肠道易位于阴道的E.coli K1株,因发生了易位才由机会致病菌转变为致病菌,产生了致病性,所以调节肠道微生态平衡,抑制致病菌的肠道粘附侵袭,成为在孕妇阶段预防本病发生的关键步骤,另外,在新生儿脑膜炎的发生过程中,E. coli K1株首先定植于新生儿肠道,之后才会穿透肠屏障和血脑屏障,引起菌血症和脑膜炎,所以预防E. coli K1株穿透肠上皮入血,也成为有效预防其引发菌血症和脑膜炎的关键环节。研究证实益生菌可预防和治疗多种感染性疾病,其机制主要包括：直接与致病微生物相互作用,通过替代、排斥和竞争机制抑制致病菌生长和粘附定植；调节肠道微生态,促进内环境共生稳态；通过空间位点和营养竞争等阻断其对肠黏膜诱导破坏和血行转移,发挥其抗感染作用；诱导结肠Mucin基因表达的上调,肠道黏膜表面粘蛋白的分泌增多,从而拮抗致病菌的粘附侵袭和跨细胞易位转移。如将益生菌应用于早期新生儿脑膜炎的预防,可克服广谱抗生素的诸多缺点。最近已有研究首次证明乳酸菌LGG能显著抑制E. coli Kl株在新生大鼠肠道粘附与入侵从而显著降低菌血症与脑膜炎发生率。但是活菌片中益生菌如何粘附于肠黏膜,是否诱导上调粘液蛋白基因表达,和如何提高肠道微生态的稳定性,目前仍不清楚。本研究的主要目的：评价三联活菌片中的益生菌对大肠埃希菌K1株在肠上皮粘附的抑制作用和预防新生儿脑膜炎的保护作用。(1)应用SYBR Green Real-time PCR法,对小鼠肠道中的益生菌-双歧杆菌和保加利亚乳杆菌,以及致病菌-E. coli K1株,进行定量检测,观察小鼠肠道益生菌的定植和对致病菌粘附的拮抗作用。(2)对益生菌和对照组小鼠肠道菌群进行变性梯度凝胶电泳图谱(DGGE)分析,观察益生菌对肠道微生态的调节作用。(3)应用活菌计数法,对乳鼠肠道、血液和脑脊液中E. coli K1株进行定量检测,观察益生菌对E. coliK1株血行转移脑膜炎的拮抗作用。(4)采用竞争性排除方法,探讨益生菌抑制E. coli K1株粘附侵袭肠上皮Lovo细胞作用。通过检测细胞培养液中乳酸脱氢酶的释放量,观察益生菌对肠上皮细胞的保护作用和E.coli K1株对细胞膜通透性的损伤作用。(5)运用RT-PCR法验证益生菌对主要粘蛋白基因MUC2表达的调节作用。益生菌能否拮抗E.coli K1株诱导的粘蛋白基因表达下降,发挥其拮抗致病菌粘附侵袭和易位作用。通过以上研究,观察活菌片中益生菌能否调节肠道微生态,初步探讨益生菌抑制致病菌的粘附、侵袭和预防血行转移入脑是否与调节肠道粘液蛋白基因表达相关。二、研究方法 1、小鼠模型观察益生菌对致病菌的粘附抑制作用提取活菌片中双歧杆菌、保加利亚乳杆菌、和E. coli K1株基因组DNA,双歧杆菌、保加利亚乳杆菌引物参照文献,E. coli K1株针对IbeA基因设计引物PCR,PCR产物克隆入pMD19-T Simple Vector载体中,转化至DH-5a宿主细胞,提取质粒倍比稀释后作标准品, 将雌性BALB/c小鼠10只,用活菌片PBS稀释液灌胃,连续14天,每天取肠道标本连续观察益生菌定殖情况。根据益生菌肠道定殖时间,将雌性BALB/c小鼠40只,随机分成四组,①益生菌组(L)、②益生菌+致病菌组(L+P)、③致病菌组(P)、④对照组(N),留取各组不同时间粪便标本。抽提其细菌DNA,进行Real-time PCR反应,观察益生菌肠道的粘附及拮抗致病菌粘附侵袭作用。 2、益生菌对肠道微生态的影响以益生菌组和对照组小鼠粪便标本总细菌为模板,以肠道总细菌16S rDNA可变区V3区为靶基因,引物序列V3-357f-GC;V3-R519,进行PCR扩增和DGGE分析。观察益生菌的肠道微生态的调节作用。 3、益生菌抑制E. coli K1株粘附、侵袭、和损伤肠上皮细胞作用参照文献采用竞争性排斥,将益生菌、E. coli K1株与Lovo细胞共孵育,检测益生菌拮抗E.coli K1株粘附与侵袭Lovo细胞的效果。设细胞只单纯孵育E.coliK1株的为对照组,计数其粘附和侵袭的菌落数,并分别计算益生菌干预下E. coliK1株粘附与侵袭的菌落数和相对黏附率、相对侵袭率。公式为相对粘附或侵袭率=(益生菌组的菌落数／对照组的菌落数)×100% 将益生菌、E. coli K1株同时和分别与Lovo细胞共孵育,通过检测细胞培养液中乳酸脱氢酶的释放量,以观察益生菌对细胞保护和细菌对细胞膜通透性的损伤作用。 4、E.coliK1株肠道血源性转移的新生大鼠模型参照文献将Sprague Dawley乳鼠随机分成益生菌组和PBS组,每组15只,分别用益生菌和PBS处理后,灌胃E. coli K1株,取肠道、血液和脑脊液标本进行定量培养,涂布于利福平抗性平板,血标本同时涂布MRS平板过夜培养并计菌落数。观察益生菌对血行转移细菌性脑膜炎的预防作用。 5、益生菌和E. coli K1株影响肠道MUC2基因表达把Sprague Dawley乳鼠随机分成益生菌组、益生菌+致病菌组、致病菌组和对照组,每组3只分别用益生菌和PBS处理3天后,给乳鼠灌胃用E. coli K1株,取肠道提取结肠组织总RNA,采用GAPDH基因做内参,用RT-PCR法观察益生菌和致病菌的诱导肠道MUC2基因表达的作用。三、研究结果 1、小鼠模型观察益生菌对致病菌的粘附抑制作用的研究结果表明,灌胃益生菌组小鼠肠道中的双歧杆菌和保加利亚乳杆菌基因拷贝数在第3天时开始明显增加,在第7天时达到稳定,且随服药天数的增加两种益生菌基因拷贝数没有明显变化；灌胃益生菌和致病菌组小鼠肠道中E. coli K1株基因拷贝数明显低于单独灌胃致病菌组。 2、变性梯度凝胶电泳图谱(DGGE)分析显示,正常BALB/c小鼠肠道细菌的多样性相似,说明用其作为动物模型对肠道菌群进行相关研究具备可行性。用非权重配对法(UPGMA)进行相似性聚类分析,提示服药后益生菌组组内个体相似性较对照组提高,说明服用益生菌可提高肠道菌群构成的一致性和肠道微生态的稳定性,可减少宿主及环境的很多因素对肠道微生态的影响。 3、益生菌抑制E. coli K1株粘附、侵袭和损伤肠上皮细胞的研究显示,益生菌能显著抑制E. coli K1株粘附Lovo细胞(P0.01),且呈剂量依赖性。益生菌对E. coli Kl株侵袭的抑制也呈剂量依赖性(P0.01)。E. coli K1株对Lovo细胞的粘附导致较高水平的LDH释出,明显高于益生菌对Lovo细胞所造成的影响,益生菌与致病菌共同粘附于Lovo细胞后,LDH的释放虽高于益生菌组,却明显低于致病菌组粘附引起的LDH的释放。 4、在E. coli K1株肠道血源性转移的新生大鼠模型中,将益生菌+致病菌组乳鼠用益生菌处理后再灌胃E.coli K1 (109CFU／只),同时设只灌胃E. coli Kl株的对照组,48 h后取肠道、血液、脑脊液标本检测。结果发现,益生菌+致病菌组乳鼠肠道定植的E.coli K1株数量显著少于对照组(P0.05),未发生菌血症,脑脊液中也未检测到E. coli K1株。对照组灌胃给予E. coli K1株(109CFU／只)后,15只乳鼠中9只出现菌血症(105 CFU/mL),2只脑脊液中出现E. coli K1株。 5、用RT-PCR法对益生菌拮抗致病菌粘附侵袭的机制进行了研究。提取各组乳鼠肠道组织总RNA,采用GAPDH基因做内参,RT-PCR法观察益生菌和致病菌诱导结肠Mucin基因的表达。结果显示灌胃益生菌组乳鼠肠道MUC2基因明显上调,而灌服E. coli K1组的MUC2基因表达则显著降低,共同灌服组基因表达无明显变化。说明益生菌诱导的MUC2基因表达上调可能成为其拮抗致病菌易位的保护机制之一。四、结论 1、活菌片中益生菌能粘附于肠黏膜,抑制大肠埃希菌K1株肠道粘附定植。 2、活菌片中益生菌可提高肠道微生态的稳定性。 3、活菌片中益生菌能显著抑制大肠埃希菌K1株对肠上皮的粘附、侵袭和损伤,以及血行转移,可能有预防细菌性脑膜炎发生的作用。 4、活菌片中益生菌可上调肠道粘液蛋白基因表达,保护肠道免受致病菌的损伤,这可能成为拮抗致病菌粘附侵袭和易位的保护机制之一。
[Abstract]:First, research background and purpose
Bacterial meningitis is the most common and most serious infection in the neonatal period of central nervous system. In the past thirty years, developed countries and developing countries, the incidence of bacterial meningitis has not changed significantly, accounting for 1 per thousand of the neonatal disease, but the mortality rate is up to 17%-38%, before the death of the deceased from the sequelae of nervous system also up to 58%.B Streptococcus pneumoniae and Escherichia coli were the most common pathogens causing neonatal meningitis, due to Streptococcus B group of pregnant women with intrapartum antibiotic selective prevention and treatment of neonatal infants, so B streptococcal infection rate decreased significantly, but also reduces the newborn of non B streptococcal infection resistance in Escherichia coli bacteria (E.coli) K1 strain is neonatal meningitis. The primary pathogen Escherichia coli strain K1 (E. coli) in 40% or 80% suffering from meningitis septicemia Children can be isolated, it is mainly due to the mother's gut, pregnant women because of their special physical fitness, intestinal colonization of E. coli K1 were easily located in the vagina, resulting in neonatal infection when they pass through the birth canal. The results show that the E. coli K1 strain 20% neonatal intestinal colonization, the intestinal colonization of the pregnant women accounted for 77%.E. coli K1 was once planted in the neonatal intestine, 0.5% intestinal metastasis can occur and blood invasive positioning, and cross the blood-brain barrier and cause meningitis.
With the extensive application of broad-spectrum antibiotics, antibiotic resistance of Escherichia coli increased, resulting in low birth weight newborns especially, very low birth weight infants of Escherichia coli meningitis mortality rate gradually increased. Antibiotic resistance has become a serious public health problem in the past ten years. The research method of complementary and alternative drugs and antibiotics it has attracted more and more attention.
The E. coli K1 strain through the intestinal barrier caused by infection of the central nervous system is still unclear, but pregnant women irritable in the vaginal E.coli K1 strain, due to the occurrence of the translocation by opportunistic bacteria into the pathogen, pathogenicity, so regulating intestinal micro ecological balance, inhibit the pathogens of intestinal adhesion and invasion. A key step in pregnant women for prevention of the disease, in addition, in the course of the occurrence of neonatal meningitis, E. coli K1 was first colonized in the neonatal intestine will penetrate the intestinal barrier and the blood brain barrier, cause bacteremia and meningitis, so to prevent E. coli K1 strain to penetrate the intestinal epithelium into the blood, also become the keys to prevent the link cause bacteremia and meningitis.
Study confirmed that probiotics can prevent and treat a variety of infectious diseases, the mechanism mainly includes: the replacement by direct interaction with pathogenic microorganisms, and the mechanism of competition, rejection caused by inhibition of growth and colonization of bacteria; regulation of intestinal microflora, promote environmental symbiotic state; through the space competition sites and nutrition on intestinal mucosal damage induced by blocking its and the blood metastasis, play its anti infection effect; induced upregulation of Mucin gene expression in the colon, increased secretion of intestinal mucosal surface mucin, invasion and metastasis of translocation across cell adhesion to antagonize pathogens. Such as the prevention of probiotics used in early neonatal meningitis, which can overcome many disadvantages of broad-spectrum antibiotics. Recent studies for the first time proof of lactic acid bacteria LGG can significantly inhibit E. coli Kl strain in intestinal adhesion and invasion in neonatal rats can significantly reduce bacteremia and meningitis. However, how the probiotics adhered to intestinal mucosa, induced mucin gene expression and how to improve the stability of intestinal microflora is still unclear.
The main purpose of this study: the protective effect of probiotics in the evaluation of triple viable for inhibition and prevention of neonatal meningitis Escherichia coli strain K1 in intestinal epithelial adhesion. (1) the application of SYBR Green Real-time PCR method of probiotics in intestinal Bifidobacterium and Lactobacillus Bulgaria, and pathogen -E. coli K1 strains were detected, observe the mice intestinal probiotic bacteria colonization and on pathogen adhesion antagonism. (2) of probiotics and denaturing gradient gel electrophoresis of control group mice intestinal flora (DGGE) analysis, adjusting Chahar probiotics on intestinal microflora. (3) using the live count method the rat intestinal bacteria, blood and cerebrospinal fluid in E. coli K1 strains were detected, to observe the antagonistic effect on the transfer of probiotic coliK1 strain E. meningitis blood line. (4) the competitive exclusion method, investigate the probiotic inhibition of E. C Oli K1 strain Lovo to intestinal epithelial cell adhesion and invasion effect. The release amount of lactate dehydrogenase in cultured cells by detecting, study the effect of probiotics on intestinal epithelial cells and the protective effect of E.coli K1 on cell membrane permeability strain injury. (5) the use of RT-PCR regulation method of probiotics on main mucin gene MUC2 expression of probiotics. Whether the mucin gene induced by antagonistic strains E.coli decreased expression of K1, play the antagonist pathogen adhesion invasion and translocation. Through the above research, observation of probiotic lactobacillus tablets can regulate the intestinal microflora, preliminary study of probiotics inhibit adhesion of pathogenic bacteria, preventing invasion and hematogenous metastasis into the brain is related to regulation of intestinal mucus protein gene expression.
Two, research methods
1, the mouse model observed the adhesion inhibition of probiotics to pathogenic bacteria
Extraction of Bifidobacterium tablets, Lactobacillus bulgaricus, and E. coli K1 genome DNA, Bifidobacterium, Lactobacillus bulgaricus primers according to the literature, the E. coli K1 strain IbeA gene primers were designed for PCR, PCR products were cloned into pMD19-T Simple Vector vector and transformed into DH-5a host cells, extracted plasmid dilution as standard goods,
10 BALB/c mice with live bacteria PBS diluted solution orally, for 14 consecutive days, every day from the intestinal colonization were continuously observed. According to the probiotic probiotic intestinal colonization time, 40 BALB/c mice were randomly divided into four groups, the probiotics group (L), the probiotics + pathogenic bacteria group (L+P), the pathogenic bacteria group (P), the control group (N), specimens of each group at different time. The extraction of fecal specimens of bacterial DNA, Real-time PCR, to observe the adhesion and antagonism of probiotics intestinal pathogen adhesion invasion.
2, the effect of probiotics on the Intestinal Microecology
In the probiotics group and control group, the total bacterial samples from stool specimens were taken as templates, and the V3 region of the total 16S rDNA variable region of the gut was used as the target gene. Primer sequence V3-357f-GC and V3-R519 were used for PCR amplification and DGGE analysis.
3, probiotics inhibit the adhesion, invasion, and damage of intestinal epithelial cells of E. coli K1 strain
According to the literature by competitive exclusion, the E. coli K1 strains of probiotics, and Lovo cells were incubated to test the effect of probiotic antagonistic E.coli strain K1 adhesion and invasion of Lovo cells. Cells only incubated E.coliK1 strain as the control group, the number of colony counts of adhesion and invasion, and don't count the colony and the relative adhesion of E. adhesion and invasion rate of coliK1 strains of probiotics, the relative rate of invasion. The formula for the relative adhesion or invasion rate (the number of CFU / = colony control group probiotic group) * 100%
Probiotics, E. coli K1 strains were incubated with Lovo cells at the same time, respectively. The release of lactate dehydrogenase from cell culture medium was observed to observe the effect of probiotics on cell protection and bacterial permeability to cell membrane.
4, a neonatal rat model of E.coliK1 strain of intestinal hematogenous metastasis
According to the literature Sprague Dawley rats were randomly divided into probiotics group and PBS group, 15 rats in each group, respectively with probiotics and PBS treatment after intragastric administration of E. coli, K1 strain from the intestinal, blood and cerebrospinal fluid specimens were cultured quantitatively, rifampicin resistant coating on the tablet, and blood samples were cultured overnight and coated MRS plate colony number. To observe the preventive effect of probiotics transfer of bacterial meningitis in blood.
5, probiotics and E. coli K1 strain influence the expression of MUC2 gene in the intestinal tract
The Sprague Dawley rats were randomly divided into group of probiotics, probiotic and pathogenic bacteria, pathogenic bacteria group and control group, 3 rats in each group were used probiotics and PBS treatment after 3 days to suckling mice with E. coli K1 strain from the intestinal extraction of total RNA in colon tissue, using GAPDH as a reference gene, and to study the effect of probiotics bacteria induced expression of intestinal MUC2 gene function by RT-PCR method.
Three, the results of the study
1, study the inhibitory effect of probiotics on model mice to observe adhesion of pathogenic bacteria showed that intragastric administration of probiotics group of intestinal Bifidobacterium and Lactobacillus gene copy number in third days began to increase significantly, to achieve stability in seventh days, and with the increase in the number of medication two kinds of probiotics have no obvious gene copy number change; copy of probiotics and pathogens of gastric irrigation in mice intestinal E. coli strain K1 gene number was significantly lower than that of gastric pathogen group.
2, denaturing gradient gel electrophoresis (DGGE) map analysis showed that the diversity of normal BALB/c mice intestinal bacteria similar to that used as the animal model to research on the intestinal microflora is feasible. Using non weight matching method (UPGMA) similarity clustering analysis, provided the individual probiotics group shown after taking the similarity higher than the control group, indicating the use of probiotics can improve the consistency of the intestinal flora and intestinal micro ecological structure stability, can reduce a lot of factors of host and environmental effects on the intestinal microflora.
3, E. coli K1 strains of probiotics inhibit adhesion, invasion and damage of intestinal epithelial cells showed that probiotics can significantly inhibit the E. coli strain K1 (P0.01), the adhesion of Lovo cells in a dose-dependent manner. The inhibition on the invasion of E. coli probiotic Kl strain was also dose dependent (P0.01) coli K1 strain on the adhesion of.E. Lovo cells leads to higher levels of LDH release was significantly higher than that caused by the effects of probiotics on Lovo cells, probiotics and pathogens to adhere to Lovo cells after the release of LDH was higher than that of the probiotic group, but significantly lower than the group of pathogenic bacteria adhesion caused by the release of LDH.
4, in E. coli K1 strains of enteric hematogenous metastasis in the neonatal rat model, probiotics + pathogenic bacteria group of neonatal rat with probiotics after intragastric administration of E.coli (109CFU / K1 only), and the control group was only coli gastric E. Kl strains, 48 h after intestinal tract, blood, cerebrospinal fluid specimens the detection results showed that probiotics + pathogenic bacteria group rat intestinal colonization of E.coli strain K1 number was significantly less than the control group (P0.05), without the occurrence of bacteremia in cerebrospinal fluid, it does not detect E. coli K1 strain. The control group was given by intragastric administration of E. coli K1 strain (109CFU / only), 15 rats in 9 only the bacteremia (105 CFU/mL), 2 E. coli K1 strain in the cerebrospinal fluid.
5, studied the antagonistic mechanism of probiotic bacteria adhesion and invasion by RT-PCR. Total RNA was extracted from rat intestinal tissue were used as a reference, the GAPDH gene expression was observed by RT-PCR of probiotics and pathogens induce colon Mucin gene. The results showed that intragastric administration of probiotics group of rat intestinal MUC2 gene was up-regulated, and Ig MUC2 E. coli K1 group gene expression was significantly reduced, the common gavage group had no obvious change in gene expression. The expression of MUC2 gene induced upregulation of probiotics may be one of the protective mechanisms against pathogen translocation.
Four. Conclusion
1, probiotics in the live bacteria tablets can adhere to the intestinal mucosa and inhibit the intestinal adhesion and colonization of Escherichia coli K1.
2, probiotics in the live bacteria tablets can improve the stability of the intestinal microecology.
3, probiotics can significantly inhibit the adhesion, invasion and damage of Escherichia coli K1 strain, and hematogenous metastasis, which may prevent bacterial meningitis.
4, probiotics can upregulate intestinal mucin gene expression and protect intestinal tract from pathogenic bacteria, which may become a protective mechanism against pathogen adhesion, invasion and translocation.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

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