柔肝固肠方改善慢性酒精性肝损伤大鼠肠道通透性的机制研究

目的：从肠道紧密连接及黏附连接角度探讨柔肝固肠方改善慢性酒精性肝损伤大鼠肠道通透性的作用机制。方法：Lieber-DeCarli酒精液体饲料饲养6周诱导大鼠慢性酒精性肝损伤模型。30只SD雄性大鼠,随机分无酒精液体饲料对照组（对照组,n＝10）和酒精液体饲料造模组（n＝20）,造模第4周将造模组大鼠随机分模型组（n＝10）,柔肝固肠方组（n＝10）,并开始灌胃给柔肝固肠方或蒸馏水直至6周末,取材前3．5 h各组以10 mg／kg的内毒素（LPS）灌胃。取材后检测：1）血清AST、ALT活性变化;2）通过门脉血浆内毒素含量测定判断小肠通透性变化;3）小肠组织电镜超微结构观察;4）小肠紧密连接蛋白ZO-1、Occludin蛋白与mRNA表达变化;5）小肠黏附连接蛋白β-Catenin和E-Cadherin蛋白表达。结果：1）柔肝固肠方可显著降低模型大鼠显著升高的AST水平（P＜0．05）;2）柔肝固肠方可降低模型大鼠显著升高的小肠通透性,柔肝固肠方组血浆内毒素含量显著低于模型组;3）电镜结果显示柔肝固肠方可显著改善Lieber-DeCarli酒精饲料喂养大鼠小肠黏膜表面微绒毛局灶性减少、变短、稀疏,排列不规则,同微绒毛相连的细胞终末网变性模糊等病理改变;4）柔肝固肠方可显著升高酒精饲料喂养肝损伤大鼠小肠组织紧密连接蛋白ZO-1、Occludin蛋白与mRNA表达及黏附连接蛋白β-Catenin和E-Cadherin蛋白表达。结论：柔肝固肠方通过改善肠上皮紧密连接及黏附连接改善慢性酒精性肝损伤大鼠肠道通透性改变。     

乳酸菌在肠道内黏附定殖的研究进展

乳酸菌是一类无芽孢的革兰氏阳性菌,能利用碳水化合物发酵产酸。由于其具有安全高效,易获取的优势,在食品、医疗及动物生产方面得到广泛运用。在动物肠道内,它利用糖类物质,生成酸性代谢物,降低肠道pH,改善肠道微生物环境。要实现乳酸菌在动物体内的良好定殖,首先要实现黏附,而黏附过程主要分为特异性结合与非特异性结合。黏附机制的相关研究中黏附素理论被普遍认可,包含了粘附蛋白学说、磷璧酸学说及细菌表面分子学说。本文就乳酸菌的黏附相关因子,体外黏附模型及黏附的影响因素做了阐述。     

骨桥蛋白13肽抑制球囊内皮剥脱术后血管狭窄的实验研究

目的：利用含有骨桥蛋白（OPN）多种功能位点的13肽（Gly^158-Lys^170）,从细胞和整体水平观察其对VSMC和单核巨噬细胞黏附、浸润以及内膜增生的影响,并初步探讨其作用机制。方法：用不同浓度OPN13肽（0,100,200,300mg／L）检测其对体外培养的平滑肌细胞（VSMc）与OPN黏附的抑制作用;以不合黏附序列的6肽分子为对照组,用以确定13肽抑制黏附特异性。用球囊内皮剥脱法建立大鼠内膜增生模型。实验动物分为4组：治疗组大鼠自术前1h及术后静脉滴注13肽,连续给药7d;对照组大鼠给予非特异性对照6肽分子;模型组大鼠给予相同剂量的生理盐水;正常对照组大鼠施假手术。并利用免疫组织化学染色和Western印迹分析方法,检测血管壁中OPN、FAK、ILK的表达变化。结果：OPN13肽能特异性的及浓度依赖性的抑制VSMC与OPN的相互作用,血管内膜剥脱后给予13肽治疗组血管壁单核／巨噬细胞浸润减少,OPN及其下游信号分子ILK,FAK表达下调,内膜增生被明显抑制。结论：含有OPN多功能位点的13肽可通过阻断OPN与膜受体的相互作用而抑制血管炎症的进展和内膜增生。     

大肠杆菌F4在3个品种猪中的黏附模式

大肠杆菌F4是引起仔猪断奶前腹泻的一种主要细菌,F4黏附于小肠上皮细胞是其致病的前提。小肠上皮细胞的F4受体是由常染色体上的基因编码的,如果无受体,仔猪表现为大肠杆菌抗性。为了研究黏附的遗传机制,本实验利用大白、长白、松辽黑猪的小肠刷状缘细胞与F4ab、F4ac、F4ad进行离体黏附实验,结果发现3品种(系)猪之间黏附情况存在显著差别(P<0.01),松辽黑猪以非黏附型为主,而长白猪中黏附型比例较高,在同一品种猪内,3种菌株的黏附比例在松辽黑猪内和大白猪内有极显著差异,但在长白猪中无显著差异。从3种细菌与刷状缘的黏附模式来看,F4ab、F4ac和F4ad分别有3种不同的受体,它们可能是由3个不同的基因座编码的。     

植物乳酸杆菌CGMCC NO．1258表层黏附蛋白同致病性大肠埃希菌竞争黏附肠上皮细胞的研究

目的了解植物乳酸杆菌CGMCC NO．1258表层黏附蛋白（IMP2）在同肠上皮细胞（Caco-2）黏附作用中的机制。方法在制备和鉴定IMP2的多克隆抗体的基础上,同EPEC进行竞争黏附肠上皮细胞中,采用体外抗体阻断的方法检测IMP2对植物乳酸菌CGMCC NO．1258黏附作用的影响。结果多克隆抗体能够有效抑制植物乳酸菌CGMCC NO．1258对肠上皮细胞的黏附,并且导致EPEC对Caco-2细胞黏附的增加。结论IMP2在植物乳酸杆菌CGMCC NO．1258同肠上皮细胞黏附中发挥着重要的作用。     

双歧杆菌黏附的研究进展

双歧杆菌是人体肠道正常菌群中的优势菌种 ,黏附和定植于肠黏膜上皮细胞后 ,对宿主发挥生物屏障、营养、免疫、抗肿瘤、抗衰老等生理作用。黏附 (ａｄｈｅｓｉｏｎ)是指微生物与宿主上皮细胞通过生物化学作用特异性地连接在一起。由于黏附是微生物与微生物、微生物与宿主相互关系的先决条件之一 ,是定植的第一步。因此 ,有关双歧杆菌黏附的研究越来越受到人们的重视。本文现就这一方面的研究进展作一综述。1　双歧杆菌对肠上皮细胞黏附的特点　　双歧杆菌进入肠道后能否黏附于宿主肠道黏膜上皮细胞表面 ,形成稳定的菌群 ,是关系到其是否能发…     

肺炎支原体对宿主细胞黏附机制的研究进展

肺炎支原体通过其末梢尖端结构即黏附细胞器与呼吸道黏膜上皮细胞受体(唾液酸共轭物或糖脂结构域)结合并定植于人体呼吸道,引起原发性非典型性肺炎等疾病。因此,其黏附机制引起了人们的重视。本文着重阐述了肺炎支原体黏附细胞器的形态、结构,黏附素与黏附辅助蛋白的种类、定位、功能及在黏附过程中的协同作用。     

QCM 监测自组装膜上心肌细胞黏附及其与心血管药物作用

目的:本试验采用石英晶体微天平(QCM)实时监测大鼠心肌细胞(H9C2)在含有细胞黏附识别多肽RGD自组装膜上的动态黏附过程及随后与两种心血管药物(一种正性肌力、另一种负性肌力)相互作用。方法:在金电极表面自组装3-巯基丙酸(MPA)单层膜,并经酰胺化共价耦合细胞黏附分子KRGD,形成对大鼠心肌细胞有特异性黏附的致密分子自组装膜。QCM以动态持续的方式实时监测MPA/RGD自组装及其不同浓度梯度H9C2细胞在自组装膜金电极上的细胞黏附过程。此外,选用20,000个H9C2细胞和正性肌力药物异丙肾上腺素、负性肌力药物维拉帕米,用QCM评估了细胞-心血管药物的相互作用。结果:与裸金电极相比,MPA/RGD修饰金电极增大了H9C2细胞黏附所引起的QCM频移(△f)与动态电阻变化(△R)响应。在所试H9C2浓度范围(5×10~4-4×10~5 cells/m L),△f与H9C2浓度呈线性关系,△R与H9C2浓度呈幂函数关系。我们用细胞粘弹性指数(CVI=△R/△f)来表征细胞的粘弹性。H9C2在异丙肾上腺素作用下,△f与△R增加、细胞-QCM表面黏附加强,细胞变硬;在维拉帕米作用下,△f与△R降低、细胞QCM表面黏附减弱,细胞变软。结论:QCM可用于不同浓度大鼠心肌细胞的动态细胞黏附监测,并可基于其细胞黏附与细胞黏弹性测定能力区分正性与负性肌力药物而可望用于心血管药物的筛选。     

金黄色葡萄球菌黏附素ClfA等抗体抑制该菌黏附牛乳腺上皮细胞效果的比较

【目的】为从免疫的角度比较和分析黏附素分子细胞外纤维蛋白原结合蛋白(Extracellular fibrinogen-binding protein,EfB)和纤维连接蛋白结合蛋A(Fibronection binding protein,FnBP)对聚集因子A(Clumping factor A,ClfA)抑制牛源金黄色葡萄球菌(Staphylococcus aureus,SA)黏附牛乳腺原代上皮细胞作用的增强效果。【方法】本试验分离培养牛乳腺上皮细胞并鉴定;将真核重组质粒EfB和FnBPA分别与ClfA组合免疫新西兰大白兔,并利用免疫后抗体体外抑制2株SA分离株侵染牛乳腺上皮细胞,采用平板计数法定量检测与比较不同免疫组合诱导抗体对黏附的抑制作用;对SA和乳腺上皮细胞分别进行荧光染色,观察不同免疫组合诱导的抗体对黏附抑制效果的差异。【结果】成功分离培养了牛乳腺原代上皮细胞;证明了构建的ClfA、FnBPA和EfB真核重组表达质粒均可在细胞中表达,且在免疫实验兔后可诱导特异性抗体的产生;细菌平板计数和荧光染色观察的结果表明,黏附素分子单独和组合免疫的抗体对该菌不同菌株(GY278和GY309)的黏附抑制能力不同,ClfA抗体的黏附抑制能力最强,FnBPA分子A区的黏附抑制能力优于D区。FnBPA、EfB分别与ClfA联合免疫抗体对黏附的抑制程度高于黏附素分子单独免疫组,FnBPA分子A区对ClfA黏附抑制的增强作用优于D区,FnBPA-A区与Efb相比对ClfA的黏附抑制增强差异不显著。【结论】FnBPA-A、FnBPA-D和EfB分别与ClfA联合免疫可不同程度地影响ClfA的黏附抑制效果,该结果为以黏附素分子为靶点的牛乳腺炎疫苗的研究提供了实验数据。     

白念珠菌对宿主的黏附机制

白念珠菌对宿主的黏附是白念珠菌感染过程的关键的第一步,因此阐明白念珠菌对宿主的黏附机制对探索新的方法预防和治疗白念珠菌感染至关重要。近年来,研究者们从白念珠菌的表面结构、黏附素以及黏附相关基因等方面对白念珠菌与宿主的黏附机制进行了大量研究。该文就白念珠菌对宿主的黏附机制进行综述。     

Factors involved in binding of Lactobacillus plantarum Lp6 to rat small intestinal mucus

AIM: To investigate the adhesion determinants of Lactobacillus plantarum Lp6, a dairy isolate. METHODS AND RESULTS: Small intestinal mucus extracted from rats was used as a substrate for adhesion. Adhesion determinants were studied by physical, chemical and enzymatic pretreatments of the bacteria, and adhesion inhibition assay. The mannose-specific adhesins were explored by studying the effect of d-mannose on adhesion and the yeast-agglutinating ability of the bacteria. It was found that adhesion decreased after bacteria were treated with sodium metaperiodate, protease K, trypsin, lithium chloride and trichloroacetic acid. However, adhesion did not decrease after trypsin-treated bacteria were incubated with cell surface protein extract. Cell surface bound exopolysaccharides were found to inhibit the adhesion. D-mannose inhibited the adhesion in a dose-dependent manner. The bacteria could significantly agglutinate yeast and lost this ability after protease K treatment. CONCLUSIONS: Adhesion was mainly mediated by the mannose specific adhesins, which might be proteins that reversibly bind to the cell surface components. Cell surface-bound exopolysaccharides were also involved in adhesion. SIGNIFICANCE AND IMPACT OF THE STUDY: The mannose-specific adhesion of Lact. plantarum Lp6 to rat mucus might be important for competing with pathogens-binding sites in gut, which may be used to resist the colonization of the pathogens.     

The adhesion of putative probiotic lactobacilli to cultured epithelial cells and porcine intestinal mucus

Aims: To investigate the adhesion of lactobacilli and their subsequent competitive exclusion ability against pathogens. Methods and Results: Four species of putative probiotic lactobacilli were studied for their adhesion abilities. First, the adhesion to Caco‐2 cells was examined by light and electron microscopy. The four species were then labelled by [methyl‐³H] thymidine and their adhesion to porcine intestinal mucus was determined by radioactivity. The tested lactobacilli showed best adhesion on ileal mucus compared with duodenal and jujenal mucus. Oxidative compound pre‐treatment (NaIO₃ and NaIO₄) dramatically decreased the adhesion of the lactobacilli to mucus. Pre‐treating mucus with proteolytic enzymes (proteinase K and trypsin) resulted in the increase of adhesion in Lactobacillus serotype Reuteri I2021, but the results in the other species were variable. Lactobacillus serotype Fermentum I5007 showed greatest adhesion potential and exerted the best competitive exclusion against Salmonella and Escherichia. Conclusions: Adhesion ability in lactobacilli is species‐specific. Lactobacilli with higher adhesion index have better competitive exclusion ability. Significance and Impact of the Study: This study suggests that there is a positive correlation between adhesion and competitive exclusion ability of lactobacilli. Additionally, the in vitro adhesion assay is a feasible way to screen unknown lactobacilli, potentially for future industrial applications.     

2种乳酸菌对牙鲆体表、消化道粘液粘附及对4种致病菌的粘附抑制性

目的 研究2株乳酸菌（L1来自牙鲆肠道,L2为干酪乳杆菌）在牙鲆体表和消化道粘液的粘附及其对4种致病菌的粘附抑制情况。方法 异硫氰酸荧光素（FITC）标记法。结果 2种乳酸菌在牙鲆体表和消化道粘液中均有粘附性。在胃的粘附效果最好,粘附百分率分别是48．18％和63．0％,L2在牙鲆体表、盲囊和肠的粘附能力强于L1。L1对白色念珠菌（Candida albicans）在各部位均无粘附抑制作用,对荧光假单胞菌（P．Fluorescens）粘附抑制作用最强．使其在牙鲆消化道粘液的粘附百分率下降了19．01％,其余依次为非01型霍乱弧菌（V．cholerae non-Оl strains）、嗜水气单胞菌（Aeromonas hydrophila）,分别降低了5．22％、3．22％;L2对非Оl型霍乱弧菌粘附抑制效果最强,使其粘附百分率降低了22．31％。使荧光假单胞菌、嗜水气单胞菌和白色念珠菌分别下降了17．50％、12．69％、5．15％。结论 对4种常见致病菌的粘附抑制在消化道粘液中好于体表。     

The effect of probiotic bacteria on the adhesion of pathogens to human intestinal mucus

Human intestinal glycoproteins extracted from faeces were used as a model for intestinal mucus to investigate adhesion of pathogenic Escherichia coli and Salmonella strains, and the effect of probiotics on this adhesion. S-fimbriated E. coli expressed relatively high adhesion in the mucus model, but the other tested pathogens adhered less effectively. Probiotic strains Lactobacillus GG and L. rhamnosus LC-705 as well as a L. rhamnosus isolated from human faeces were able to slightly reduce S-fimbria-mediated adhesion. Adhesion of S. typhimurium was significantly inhibited by probiotic L. johnsonii LJ1 and L. casei Shirota. Lactobacillus GG and L. rhamnosus (human isolate) increased the adhesion of S. typhimurium suggesting that the pathogen interacts with the probiotic.     

乳杆菌表面粘附蛋白的提取、鉴定及粘附特异性的初步研究

以从健康牙鲆肠道中分离筛选的乳杆菌L15(Lactobacillussp.L15)和嗜酸乳杆菌ATCC4356为实验材料,应用5mol/L LiCl提取其表面蛋白,利用蛋白印迹法鉴定出在L15表面蛋白中分子量为61.8kDa和54.6kDa的蛋白质分别参与对牙鲆和鲤鱼粘液的粘附过程,为新发现的粘附蛋白种类,将其命名为MAPPpo1和MAPPcc。ATCC4356中分子量分别为43.0kDa和63.3kDa的两个表面蛋白参与对牙鲆粘液的粘附,而分子量为43.0kDa的蛋白参与对鲤鱼粘液的粘附。同时,蛋白质印迹法显示,L15和ATCC4356在牙鲆和鲤鱼肠粘液中均具有相同的粘附受体,在牙鲆肠粘液中是分子量为29.7kDa和30.3kDa的两种蛋白质,而在鲤鱼肠粘液中只有分子量为26.2kDa的蛋白作为受体参与L15和ATCC4356的粘附过程。结果显示,乳杆菌对肠粘液的粘附不但具有菌种的特异性,而且也有宿主的特异性。     

A Mucus Adhesion Promoting Protein,MapA, Mediates the Adhesion of Lactobacillus reuteri to Caco-2 Human Intestinal Epithelial Cells

Lactobacillus reuteri is one of the dominant lactobacilli found in the gastrointestinal tract of various animals. A surface protein of L. reuteri 104R, mucus adhesion promoting protein (MapA), is considered to be an adhesion factor of this strain. We investigated the relation between MapA and adhesion of L. reuteri to human intestinal (Caco-2) cells. Quantitative analysis of the adhesion of L. reuteri strains to Caco-2 cells showed that various L. reuteri strains bind not only to mucus but also to intestinal epithelial cells. In addition, purified MapA bound to Caco-2 cells, and this binding inhibited the adhesion of L. reuteri in a concentration-dependent manner. Based on these observations, the adhesion of L. reuteri appears due to the binding of MapA to receptor-like molecules on Caco-2 cells. Further, far-western analysis indicated the existence of multiple receptor-like molecules in Caco-2 cells.     

Role of commercial probiotic strains against human pathogen adhesion to intestinal mucus

AIMS: The aims of this study present were to assess and to evaluate in vitro the abilities of commercial probiotic strains derived from fermented milk products and related sources currently marketed in European countries, to inhibit, compete and displace the adhesion of selected potential pathogens to immobilized human mucus. METHODS AND RESULTS: The adhesion was assessed by measuring the radioactivity of bacteria adhered to the human mucus. We tested 12 probiotic strains against eight selected pathogens. All strains tested were able to adhere to mucus. All probiotic strains tested were able to inhibit and displace (P<0.05) the adhesion of Bacteroides, Clostridium, Staphylococcus and Enterobacter. In addition, the abilities to inhibit and to displace adhered pathogens depended on both the probiotic and the pathogen strains tested suggesting that several complementary mechanisms are implied in the processes. CONCLUSIONS: Our results indicate the need for a case-by-case assessment in order to select strains with the ability to inhibit or displace a specific pathogen. Probiotics could be useful to correct deviations observed in intestinal microbiota associated with specific diseases and also, to prevent pathogen infections. SIGNIFICANCE AND IMPACT OF THE STUDY: The competitive exclusion properties of probiotics as well as their ability to displace and inhibit pathogens are the most importance for therapeutic manipulation of the enteric microbiota. The application of such strategies could contribute to expand the beneficial properties on human health against pathogen infection.     

Modulation of anti-inflammatory response in lipopolysaccharide stimulated human THP-1 cell line and mouse model at gene expression level with indigenous putative probiotic lactobacilli

The anti-inflammatory potential of eight indigenous probiotic Lactobacillus isolates was evaluated in vitro in terms of modulating the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in human acute monocytic leukemia (THP-1) cells under inflammatory conditions. Amongst these, Lactobacillus plantarum Lp91 was the most potent anti-inflammatory strain as it evoked a significant (P < 0.001) down-regulation of TNF-α by −1.45-fold relative to the control in THP-1 cells. However, in terms of IL-6 expression, all the strains could up-regulate its expression considerably at different levels. Hence, based on in vitro expression of TNF-α, Lp91 was selected for in vivo study in lipopolysaccharide (LPS)-induced mouse model to look at the expression of TNF-α, IL-6, monocyte chemotactic protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule (ICAM-1) and E-selectin in mouse aorta. In LPS challenged (2 h) mice group fed with Lp91 for 10 days, TNF-α, IL-6, MCP-1, VCAM-1, ICAM-1 and E-selectin expressions were significantly down-regulated by 3.10-, 10.02-, 4.22-, −3.14-, 2.28- and 5.71-fold relative to control conditions. In conclusion, Lp91 could serve as a candidate probiotic strain to explore it as a possible biotherapeutic anti-inflammatory agent against inflammatory diseases including cardiovascular disease.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-013-0347-5) contains supplementary material, which is available to authorized users.     

Adhesion of probiotic lactobacilli to chick intestinal mucus

In the present work, interactions between three Lactobacillus strains (Lactobacillus fermentum CRL1015, Lactobacillus animalis CRL1014, and Lactobacillus fermentum CRL1016) and chicken small intestinal mucus were determined. Three lactobacilli isolated from chicken and selected by their potentially probiotic properties were able to grow in mucus preparations. Three peaks from gel filtration chromatography of intestinal mucus were obtained. The adhesion to three mucus fractions (I, II, and III), especially fraction III, was higher (P < 0.01) in L. fermentum CRL1015 than L. animalis CRL1014. Pretreatment of this fraction with proteases and metaperiodate showed lower (P < 0.01) adhesion values than that of the control, suggesting that a glycoprotein from the mucus acts as a receptor for L. fermentum CRL1015. Highest adhesion values were obtained at pH 7 and 42 degrees C, and neither the removal of divalent cations with ethylenediaminetetraacetic acid (EDTA) nor the addition of calcium produced significant variation from the adhesion values of the control (P > 0.01). This adhesion was only inhibited by N-acetyl-glucosamine. Salmonella pullorum and Salmonella gallinarum showed high (P < 0.01) values of adhesion to chick intestinal mucus. The results obtained from assays of the inhibition of adherence of Salmonella spp. to mucus, immobilized in polystyrene tissue culture wells, indicated that the pathogen adhesion was not reduced by lactobacilli (P > 0.05) or their spent culture supernatants (P > 0.05), suggesting that these strains did not interfere with the binding sites for Salmonella spp. adhesion to the small intestinal mucus.     

Quantitative evaluation of adhesion of lactobacilli isolated from human intestinal tissues to human colonic mucin using surface plasmon resonance (BIACORE assay)

AIMS: To isolate lactobacilli from the mucus layer of the human intestine and evaluate their adhesion abilities using a BIACORE assay. METHODS AND RESULTS: Thirty strains of lactobacilli were isolated from the mucus layer of normal human intestinal tissues using conventional plate culture. The strains were identified using homology comparisons of the 16S rDNA sequence to databases as Lactobacillus salivarius (26%), Lactobacillus fermentum (13%), Lactobacillus gasseri (10%), Lactobacillus paracasei (7%), Lactobacillus casei (3%), Lactobacillus mucosae (3%) and Lactobacillus plantarum (3%). Lactobacillus plantarum LA 318 shows the highest adhesion to human colonic mucin (HCM) using the BIACORE assay at 115.30 +/- 12.37 resonance unit (RU). The adhesion of cell wall surface proteins from strain LA 318 was significantly higher to HCM than to bovine serum albumin (BSA; P < 0.05). CONCLUSIONS: We isolated 30 strains of lactobacilli. Lactobacillus salivarius was the predominant species of lactobacilli isolated in this study. The adhesion of strain LA 318 isolated from human transverse colon to its mucin was shown. The adhesion could be mediated by lectin-like components on the bacterial cell surface. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study where lactobacilli were isolated from human intestinal tissues and shown to adhere to HCM.