本文選題：益生菌　切入點(diǎn)：大腸埃希菌K1株　出處：《南方醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 一、研究背景和目的 細(xì)菌性腦膜炎是新生兒時(shí)期中樞神經(jīng)系統(tǒng)最常見(jiàn)最嚴(yán)重的感染。在過(guò)去三十年中,發(fā)達(dá)國(guó)家和發(fā)展中國(guó)家,細(xì)菌性腦膜炎發(fā)病率一直都沒(méi)有明顯變化,占新生兒疾病的1‰,但死亡率達(dá)17%-38%,未死亡者遺留神經(jīng)系統(tǒng)后遺癥者也高達(dá)58%。B族鏈球菌和大腸埃希菌是引起新生兒細(xì)菌性腦膜炎的最常見(jiàn)病原體,由于對(duì)B族鏈球菌攜帶孕婦的產(chǎn)時(shí)預(yù)防和對(duì)新生兒的抗生素選擇性治療,所以新生兒B族鏈球菌感染率明顯減低,但同時(shí)也降低了新生兒對(duì)非B族鏈球菌感染的抵抗力,致使大腸埃希菌(E.coli)K1株成為目前新生兒細(xì)菌性腦膜炎的首要致病菌。大腸埃希菌(E. coli)K1株在40%敗血癥或80%腦膜炎患兒中都可分離到,它主要源于母親腸道,孕婦因其特殊體質(zhì),腸道定殖的E. coli K1株容易易位于陰道,致使新生兒在通過(guò)產(chǎn)道時(shí)獲得感染。研究表明新生兒20%腸道中有E. coli K1株定植,其中孕婦腸道有該菌定植的占77%。E. coli K1株一旦定植于新生兒腸道,0.5%可發(fā)生腸道侵襲性定位轉(zhuǎn)移而入血,并穿過(guò)血腦屏障而引發(fā)腦膜炎。 隨著廣譜抗生素的廣泛應(yīng)用,耐藥性大腸埃希菌不斷增多,致使新生兒尤其是低出生體重、極低出生體重兒的大腸埃希菌性腦膜炎病死率逐漸增高。抗生素耐藥性已成為過(guò)去十年中一個(gè)嚴(yán)重的公共健康問(wèn)題。補(bǔ)充和替代抗生素治療的藥物和方法研究也越來(lái)越受到關(guān)注。 目前E. coli K1株如何通過(guò)腸屏障造成中樞神經(jīng)系統(tǒng)感染,仍不清楚,但孕婦腸道易位于陰道的E.coli K1株,因發(fā)生了易位才由機(jī)會(huì)致病菌轉(zhuǎn)變?yōu)橹虏【?產(chǎn)生了致病性,所以調(diào)節(jié)腸道微生態(tài)平衡,抑制致病菌的腸道粘附侵襲,成為在孕婦階段預(yù)防本病發(fā)生的關(guān)鍵步驟,另外,在新生兒腦膜炎的發(fā)生過(guò)程中,E. coli K1株首先定植于新生兒腸道,之后才會(huì)穿透腸屏障和血腦屏障,引起菌血癥和腦膜炎,所以預(yù)防E. coli K1株穿透腸上皮入血,也成為有效預(yù)防其引發(fā)菌血癥和腦膜炎的關(guān)鍵環(huán)節(jié)。 研究證實(shí)益生菌可預(yù)防和治療多種感染性疾病,其機(jī)制主要包括：直接與致病微生物相互作用,通過(guò)替代、排斥和競(jìng)爭(zhēng)機(jī)制抑制致病菌生長(zhǎng)和粘附定植；調(diào)節(jié)腸道微生態(tài),促進(jìn)內(nèi)環(huán)境共生穩(wěn)態(tài)；通過(guò)空間位點(diǎn)和營(yíng)養(yǎng)競(jìng)爭(zhēng)等阻斷其對(duì)腸黏膜誘導(dǎo)破壞和血行轉(zhuǎn)移,發(fā)揮其抗感染作用；誘導(dǎo)結(jié)腸Mucin基因表達(dá)的上調(diào),腸道黏膜表面粘蛋白的分泌增多,從而拮抗致病菌的粘附侵襲和跨細(xì)胞易位轉(zhuǎn)移。如將益生菌應(yīng)用于早期新生兒腦膜炎的預(yù)防,可克服廣譜抗生素的諸多缺點(diǎn)。最近已有研究首次證明乳酸菌LGG能顯著抑制E. coli Kl株在新生大鼠腸道粘附與入侵從而顯著降低菌血癥與腦膜炎發(fā)生率。但是活菌片中益生菌如何粘附于腸黏膜,是否誘導(dǎo)上調(diào)粘液蛋白基因表達(dá),和如何提高腸道微生態(tài)的穩(wěn)定性,目前仍不清楚。 本研究的主要目的：評(píng)價(jià)三聯(lián)活菌片中的益生菌對(duì)大腸埃希菌K1株在腸上皮粘附的抑制作用和預(yù)防新生兒腦膜炎的保護(hù)作用。(1)應(yīng)用SYBR Green Real-time PCR法,對(duì)小鼠腸道中的益生菌-雙歧桿菌和保加利亞乳桿菌,以及致病菌-E. coli K1株,進(jìn)行定量檢測(cè),觀察小鼠腸道益生菌的定植和對(duì)致病菌粘附的拮抗作用。(2)對(duì)益生菌和對(duì)照組小鼠腸道菌群進(jìn)行變性梯度凝膠電泳圖譜(DGGE)分析,觀察益生菌對(duì)腸道微生態(tài)的調(diào)節(jié)作用。(3)應(yīng)用活菌計(jì)數(shù)法,對(duì)乳鼠腸道、血液和腦脊液中E. coli K1株進(jìn)行定量檢測(cè),觀察益生菌對(duì)E. coliK1株血行轉(zhuǎn)移腦膜炎的拮抗作用。(4)采用競(jìng)爭(zhēng)性排除方法,探討益生菌抑制E. coli K1株粘附侵襲腸上皮Lovo細(xì)胞作用。通過(guò)檢測(cè)細(xì)胞培養(yǎng)液中乳酸脫氫酶的釋放量,觀察益生菌對(duì)腸上皮細(xì)胞的保護(hù)作用和E.coli K1株對(duì)細(xì)胞膜通透性的損傷作用。(5)運(yùn)用RT-PCR法驗(yàn)證益生菌對(duì)主要粘蛋白基因MUC2表達(dá)的調(diào)節(jié)作用。益生菌能否拮抗E.coli K1株誘導(dǎo)的粘蛋白基因表達(dá)下降,發(fā)揮其拮抗致病菌粘附侵襲和易位作用。通過(guò)以上研究,觀察活菌片中益生菌能否調(diào)節(jié)腸道微生態(tài),初步探討益生菌抑制致病菌的粘附、侵襲和預(yù)防血行轉(zhuǎn)移入腦是否與調(diào)節(jié)腸道粘液蛋白基因表達(dá)相關(guān)。 二、研究方法 1、小鼠模型觀察益生菌對(duì)致病菌的粘附抑制作用 提取活菌片中雙歧桿菌、保加利亞乳桿菌、和E. coli K1株基因組DNA,雙歧桿菌、保加利亞乳桿菌引物參照文獻(xiàn),E. coli K1株針對(duì)IbeA基因設(shè)計(jì)引物PCR,PCR產(chǎn)物克隆入pMD19-T Simple Vector載體中,轉(zhuǎn)化至DH-5a宿主細(xì)胞,提取質(zhì)粒倍比稀釋后作標(biāo)準(zhǔn)品, 將雌性BALB/c小鼠10只,用活菌片PBS稀釋液灌胃,連續(xù)14天,每天取腸道標(biāo)本連續(xù)觀察益生菌定殖情況。根據(jù)益生菌腸道定殖時(shí)間,將雌性BALB/c小鼠40只,隨機(jī)分成四組,①益生菌組(L)、②益生菌+致病菌組(L+P)、③致病菌組(P)、④對(duì)照組(N),留取各組不同時(shí)間糞便標(biāo)本。抽提其細(xì)菌DNA,進(jìn)行Real-time PCR反應(yīng),觀察益生菌腸道的粘附及拮抗致病菌粘附侵襲作用。 2、益生菌對(duì)腸道微生態(tài)的影響 以益生菌組和對(duì)照組小鼠糞便標(biāo)本總細(xì)菌為模板,以腸道總細(xì)菌16S rDNA可變區(qū)V3區(qū)為靶基因,引物序列V3-357f-GC;V3-R519,進(jìn)行PCR擴(kuò)增和DGGE分析。觀察益生菌的腸道微生態(tài)的調(diào)節(jié)作用。 3、益生菌抑制E. coli K1株粘附、侵襲、和損傷腸上皮細(xì)胞作用 參照文獻(xiàn)采用競(jìng)爭(zhēng)性排斥,將益生菌、E. coli K1株與Lovo細(xì)胞共孵育,檢測(cè)益生菌拮抗E.coli K1株粘附與侵襲Lovo細(xì)胞的效果。設(shè)細(xì)胞只單純孵育E.coliK1株的為對(duì)照組,計(jì)數(shù)其粘附和侵襲的菌落數(shù),并分別計(jì)算益生菌干預(yù)下E. coliK1株粘附與侵襲的菌落數(shù)和相對(duì)黏附率、相對(duì)侵襲率。公式為相對(duì)粘附或侵襲率=(益生菌組的菌落數(shù)／對(duì)照組的菌落數(shù))×100% 將益生菌、E. coli K1株同時(shí)和分別與Lovo細(xì)胞共孵育,通過(guò)檢測(cè)細(xì)胞培養(yǎng)液中乳酸脫氫酶的釋放量,以觀察益生菌對(duì)細(xì)胞保護(hù)和細(xì)菌對(duì)細(xì)胞膜通透性的損傷作用。 4、E.coliK1株腸道血源性轉(zhuǎn)移的新生大鼠模型 參照文獻(xiàn)將Sprague Dawley乳鼠隨機(jī)分成益生菌組和PBS組,每組15只,分別用益生菌和PBS處理后,灌胃E. coli K1株,取腸道、血液和腦脊液標(biāo)本進(jìn)行定量培養(yǎng),涂布于利福平抗性平板,血標(biāo)本同時(shí)涂布MRS平板過(guò)夜培養(yǎng)并計(jì)菌落數(shù)。觀察益生菌對(duì)血行轉(zhuǎn)移細(xì)菌性腦膜炎的預(yù)防作用。 5、益生菌和E. coli K1株影響腸道MUC2基因表達(dá) 把Sprague Dawley乳鼠隨機(jī)分成益生菌組、益生菌+致病菌組、致病菌組和對(duì)照組,每組3只分別用益生菌和PBS處理3天后,給乳鼠灌胃用E. coli K1株,取腸道提取結(jié)腸組織總RNA,采用GAPDH基因做內(nèi)參,用RT-PCR法觀察益生菌和致病菌的誘導(dǎo)腸道MUC2基因表達(dá)的作用。 三、研究結(jié)果 1、小鼠模型觀察益生菌對(duì)致病菌的粘附抑制作用的研究結(jié)果表明,灌胃益生菌組小鼠腸道中的雙歧桿菌和保加利亞乳桿菌基因拷貝數(shù)在第3天時(shí)開(kāi)始明顯增加,在第7天時(shí)達(dá)到穩(wěn)定,且隨服藥天數(shù)的增加兩種益生菌基因拷貝數(shù)沒(méi)有明顯變化；灌胃益生菌和致病菌組小鼠腸道中E. coli K1株基因拷貝數(shù)明顯低于單獨(dú)灌胃致病菌組。 2、變性梯度凝膠電泳圖譜(DGGE)分析顯示,正常BALB/c小鼠腸道細(xì)菌的多樣性相似,說(shuō)明用其作為動(dòng)物模型對(duì)腸道菌群進(jìn)行相關(guān)研究具備可行性。用非權(quán)重配對(duì)法(UPGMA)進(jìn)行相似性聚類(lèi)分析,提示服藥后益生菌組組內(nèi)個(gè)體相似性較對(duì)照組提高,說(shuō)明服用益生菌可提高腸道菌群構(gòu)成的一致性和腸道微生態(tài)的穩(wěn)定性,可減少宿主及環(huán)境的很多因素對(duì)腸道微生態(tài)的影響。 3、益生菌抑制E. coli K1株粘附、侵襲和損傷腸上皮細(xì)胞的研究顯示,益生菌能顯著抑制E. coli K1株粘附Lovo細(xì)胞(P0.01),且呈劑量依賴(lài)性。益生菌對(duì)E. coli Kl株侵襲的抑制也呈劑量依賴(lài)性(P0.01)。E. coli K1株對(duì)Lovo細(xì)胞的粘附導(dǎo)致較高水平的LDH釋出,明顯高于益生菌對(duì)Lovo細(xì)胞所造成的影響,益生菌與致病菌共同粘附于Lovo細(xì)胞后,LDH的釋放雖高于益生菌組,卻明顯低于致病菌組粘附引起的LDH的釋放。 4、在E. coli K1株腸道血源性轉(zhuǎn)移的新生大鼠模型中,將益生菌+致病菌組乳鼠用益生菌處理后再灌胃E.coli K1 (109CFU／只),同時(shí)設(shè)只灌胃E. coli Kl株的對(duì)照組,48 h后取腸道、血液、腦脊液標(biāo)本檢測(cè)。結(jié)果發(fā)現(xiàn),益生菌+致病菌組乳鼠腸道定植的E.coli K1株數(shù)量顯著少于對(duì)照組(P0.05),未發(fā)生菌血癥,腦脊液中也未檢測(cè)到E. coli K1株。對(duì)照組灌胃給予E. coli K1株(109CFU／只)后,15只乳鼠中9只出現(xiàn)菌血癥(105 CFU/mL),2只腦脊液中出現(xiàn)E. coli K1株。 5、用RT-PCR法對(duì)益生菌拮抗致病菌粘附侵襲的機(jī)制進(jìn)行了研究。提取各組乳鼠腸道組織總RNA,采用GAPDH基因做內(nèi)參,RT-PCR法觀察益生菌和致病菌誘導(dǎo)結(jié)腸Mucin基因的表達(dá)。結(jié)果顯示灌胃益生菌組乳鼠腸道MUC2基因明顯上調(diào),而灌服E. coli K1組的MUC2基因表達(dá)則顯著降低,共同灌服組基因表達(dá)無(wú)明顯變化。說(shuō)明益生菌誘導(dǎo)的MUC2基因表達(dá)上調(diào)可能成為其拮抗致病菌易位的保護(hù)機(jī)制之一。 四、結(jié)論 1、活菌片中益生菌能粘附于腸黏膜,抑制大腸埃希菌K1株腸道粘附定植。 2、活菌片中益生菌可提高腸道微生態(tài)的穩(wěn)定性。 3、活菌片中益生菌能顯著抑制大腸埃希菌K1株對(duì)腸上皮的粘附、侵襲和損傷,以及血行轉(zhuǎn)移,可能有預(yù)防細(xì)菌性腦膜炎發(fā)生的作用。 4、活菌片中益生菌可上調(diào)腸道粘液蛋白基因表達(dá),保護(hù)腸道免受致病菌的損傷,這可能成為拮抗致病菌粘附侵襲和易位的保護(hù)機(jī)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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R378

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