【作者】 王斌；

【导师】 魏泓；

【作者基本信息】 第三军医大学 ， 细胞生物学， 2005， 博士

【摘要】 益生菌是人体肠道内正常的生理性有益菌,是肠粘膜生物屏障的主要组成部分,进入肠道内的益生菌通过粘附定植于肠粘膜表面以保护肠粘膜上皮细胞免受各种病原微生物的损伤,所以益生菌在肠道内的粘附定植是其发挥生理作用的前提和基础,但是,目前对于益生菌粘附性能的检测还没有一个合适的实验方法;而且,益生菌的粘附性能具有菌株特异性,高粘附性能的模式菌株较少,有关粘附的生物学保护作用和粘附机制等研究尚缺乏系统性资料。因此,本研究通过优化体外粘附实验方法,对11株益生菌的粘附性能进行检测,筛选得到粘附性能较好的乳杆菌,以该菌株作为研究材料,对其粘附的生物学保护作用进行研究,并对其参与粘附的粘附素分子进行初步的提取和鉴定。方法:(1)以体外培养的肠上皮样细胞HT-29为模型,对影响益生菌体外粘附细胞的外部因素进行了探讨,通过优化的体外细胞粘附实验方法,对11株益生菌的粘附性能进行检测,并与放射性同位素标记法和粘蛋白的ELISA法进行比较。通过制备粘附性能较好菌株的多克隆抗体,采用改进的Carnoy固定液对肠粘膜组织标记进行固定,然后制备冰冻切片并进行免疫组化分析,建立乳杆菌体内粘附定植模型。(2)比较不同粘附性能的菌株对体外培养的HT-29细胞的保护作用。建立抗生素脱污染EPEC感染的小鼠模型,通过肠粘膜病理切片、肠道菌群分析以及细菌移位分析等,观察乳杆菌对肠粘膜屏障的保护作用。(3)对筛选得到的高低粘附力菌株的细菌表面成分和培养乏液进行酶、热、过碘酸钠等处理,以观察细菌表面成分和培养乏液对粘附的影响;采用溶菌酶、变溶菌素以及硫酸铵等对细菌胞壁表面蛋白和培养乏液中的蛋白进行提取,通过与HRP标记的粘蛋白受体和NHS-Biotin标记的HT-29进行杂交,对参与粘附的细菌胞壁表面蛋白和促粘附因子进行初步鉴定;通过制备细菌的多克隆抗体,筛选与HT-29细胞粘附的胞壁靶蛋白;并将粘附相关蛋白进行质谱分析。结果:(1)优化了体外粘附评价实验方法。pH值、孵育时间、菌液浓度以及菌的生长状态均可影响乳杆菌与HT-29细胞的粘附。优化其基本步骤为:采用培养14天后的肠上皮样细胞HT-29,与培养至稳定期的菌液浓度为1～2×10~8/ml益生菌(含益生菌的SCS),在37℃下共孵育60min,然后用灭菌的PBS洗涤细胞5次,甲醇固定20min,革兰氏染色,镜检计数。11株益生菌的粘附性能检测结果表明,乳杆菌属和双歧杆菌属的各种间不同菌株在体外对HT-29细胞的粘附能力相差很大,其中罗伊氏乳杆菌JCM1081的粘附指数为495.07±80.03,显著高于其它益生菌株;嗜酸乳杆菌La1.1878和长双歧杆菌B050102-14的粘附能力较弱,其粘附指数分别为135.43±13.93和47.17±5.84;制备了罗伊氏乳杆菌JCM1081的多克隆抗体,免疫组化分析表明,在小鼠肠组织中可见罗伊氏乳杆菌JCM1081在肠粘膜表面呈弥散状分布、定植,形成肠粘膜菌群生物屏障;(2)粘附性能较好的罗伊氏乳杆菌JCM1081在体外可抑制致病性大肠杆菌对肠上皮样细胞HT-29的粘附(28.1%)(P＜0.01)和侵袭(23.9%)(P＜0.01),粘附力较弱的嗜酸乳杆菌La1.1878对致病性大肠杆菌的粘附抑制率为12.4%(P＜0.01),侵袭抑制率为7.5%(P＜0.01)。抑菌实验结果显示,嗜酸乳杆菌La1.1878对EPEC的抑制作用显著高于罗伊氏乳杆菌JCM1081(P＜0.01)。乳杆菌粘附肠上皮样细胞HT-29后,细胞结构正常,乳酸脱氢酶释放量和细胞活力无明显变化。不显著影响单层细胞的通透性和细胞微丝骨架F-actin。乳杆菌减弱了致病性大肠杆菌对肠上皮细胞样HT-29的损伤(P＜0.05)。小鼠体内实验结果表明,灌胃乳杆菌的治疗组肠道菌群中双歧杆菌和乳杆菌的数量与致病性大肠杆菌感染的模型组相比,有显著增加(P＜0.05);在肝、脾、肾等实质器官以及肠系膜淋巴结处发现有部分肠杆菌,但数量明显低于EPEC感染模型组(P＜0.01)。(3)罗伊氏乳杆菌JCM1081菌体经胰蛋白酶、蛋白酶K处理后,其对HT-29细胞的粘附力显著下降(P＜0.01);提取了罗伊氏乳杆菌JCM1081细胞壁表面蛋白,Westernblot结果显示29kD和14kD的两种蛋白与粘蛋白受体和HT-29细胞的杂交中都出现了强阳性;多抗鉴定结果显示,29kD的蛋白与罗伊氏乳杆菌JCM1081的多克隆抗体产生杂交阳性条带;将29kD的蛋白进行质谱分析后,结果表明,此蛋白与罗伊氏乳杆菌ATCC55730的lr0793蛋白的相似性高达71.1%,实际分子量为28519.54Da,有263个氨基酸,PI为9.78,属于ATP-Binding Cassette蛋白家族。采用相同的方法对其它8株乳杆菌的胞壁表面蛋白进行了鉴定,结果显示,其中,只有鼠李糖乳杆菌1.120存在29kD的杂交阳性条带,而嗜酸乳杆菌L050103-12存在43kD、63kD以及85kD的三种蛋白杂交阳性,在其它乳杆菌中未检测出杂交阳性条带。将罗伊氏乳杆菌JCM1081的培养乏液用PBS、新鲜的MRS替换,或经过胰蛋白酶或蛋白酶K处理,乳杆菌JCM1081对HT-29的粘附力显著下降(P＜0.01);罗伊氏乳杆菌JCM1081的SCS不能促进低粘附力菌株嗜酸乳杆菌La1.1878对HT-29细胞的粘附;对JCM1081的SCS进行了蛋白提取,经初步提纯,SDS-PGAE和Western blol结果表明,在其SCS中存在一种29kD的小分子蛋白,与HRP标记的粘蛋白受体杂交可产生阳性。结论:1.细胞培养革兰氏染色法是一种有效的检测益生菌粘附性能的实验方法,它与放射性同位素法、荧光标记法以及活菌计数法相比较,具有操作简单,重复性好,实验结果稳定可靠等优点。2.乳杆菌属和双歧杆菌属的各种间不同菌株粘附能力相差很大,筛选了高粘附力菌株罗伊氏乳杆菌JCM1081,为研究乳杆菌的粘附保护作用及粘附机制提供了良好模式材料,同时也提供了一株高粘附性能的益生菌株。3.高粘附力菌株罗伊氏乳杆菌JCM1081对HT-29细胞具有粘附保护作用,其粘附性能的高低与生物保护作用的强弱具有一定相关性。乳杆菌JCM1081可抑制致病性大肠杆菌对肠上皮样细胞HT-29的粘附和侵袭,减弱了致病性大肠杆菌对肠上皮细胞样HT-29的损伤;罗伊氏乳杆菌JCM1081可粘附定植于肠粘膜,形成生物屏障,维持并保护肠粘膜的正常功能。4.JCM1081的粘附能力与其细胞壁表面的蛋白成分密切相关。分离纯化鉴定了其中的一种重要的参与其粘附的新蛋白成分,该蛋白分子量为29kDa,是其主要的粘附素分子之一;29kD蛋白与罗伊氏乳杆菌ATCC55730的lr0793蛋白相似性高达71.1%,有263个氨基酸,PI为9.78,属于ATP-Binding Cassette蛋白家族。其培养乏液中存在有29kD的促粘附因子,与胞壁表面29kD的粘附素蛋白可能为同一类物质。该结果提示29kD蛋白为一种分泌性蛋白,一部分分布于细胞壁表面,用于识别细胞膜表面的粘附素受体,另一部分则分泌至胞外培养乏液中,介导了菌体与细胞的粘附。29kD的粘附素蛋白在乳杆菌中并不是普遍存在的,不同种属的乳杆菌,其参与粘附的蛋白种类亦存在多样性。更多还原

【Abstract】 Probiotics play an important role in controlling undesirable microflora in the gastrointestinal tract of humans and animals.They have been shown to possess inhibitory activity toward the growth of pathogenic bacteria.The characteristics of adhesion of probiotics to the intestinal mucosal surface are a critical prerequisite for exerting beneficial effect to their host organisms.But there have some difficulties involved in studying bacterial adhesion in vivo,especially in humans.To date,there have no a proper evaluation method for adhesion in vitro,and little is known about the mechanisms by which is might occour in the human gastrointestinal tract and these bacteria attach to the intestinal mucosa. Therefore,which have led to the development of in vitro model systems for the preliminary selection of potentially adherent strains,and to gain further understanding of the mechanisms by which lactobacilli adhere to human intestinal cells and investigate intestinal mucosal barrier to prevent the infection of the enteropathogenic bacteria.Methods:1.In vitro model of HT-29 cell culture was employed to investigate the influence of the pH,buffer,growth phase,incubation time and bacteria concentration on adherence of Lactobacillus.A critical validation of in vitro adhesion model was used to screen and assess adhesive properties of 11 strains of probiotics.Methods of radioactivity and ELISA of mucin were compared as complement.Anti-JCM1081 polyclonal antibodies were prepared by immunizing rabbit to investigate colonization of Lactobacillus JCM1081 on intestinal mucosa through frozen section and immunohistochemistry.2.Different adhesive properties of Lactobacillus strains were compared to investigate the beneficial effect on protection of intestinal epithelial cells.Antibiotics decontaminated and EPEC infected mice model was established and HE stained slide,gut microflora analysis and bacteria translocation were observed for intestinal mucosal changes.3.Lactobacillus with spent culture supernatant were subjected to various treatments including heat shock,trypsin, pronase and chemical products to study on factors involved in adherence.Extraction of the cell wall surface proteins with lysozyme and mutanolysin were performed and spent culture supernatant was fractionated by ammonium sulfate precipitation.The proteins which associated with adhesion of Lactobacillus to HT-29 and mucin were dectected in the fractions by SDS-PAGE and Western blotting with horseradish peroxidase labeled mucin and NHS-Biotin labeled HT-29 cells and the adhesin of Lactobacilli were dectected in Western blotting with Anti-JCM1081 polyclonal antibodies.The proteins which asscoiated with adhesion of Lactobacillus to HT-29 cells were purified to mass spectrographic analysis.Results:1.An evaluation method for adhesion was optimized.The pH,bacteria concentration and incubation time,growth phase of Lactobacilli have influence on adhesion of Lactobacilli to HT-29 cells.In general,the adhesion approach was optimized,and the procedure is that the HT-29 monolayers which cultured at post confluence after 15d were washed twice with PBS,1 ml 1～2×10⁸ bacteria（with spent culture supernatant） suspension was mixed with DMEM and then incubated at 37℃in 10%CO₂/90%air.After incubation, the monolayers were washed five times with sterile PBS,fixed with methanol, Gram-stained,and examined microscopically.Eleven strains were examined for their ability to adhere to cultured HT-29 cells by Gram stain and radioactivity methods.The ability of the Lactobacilli and Bifidobacteria to adhere to the HT-29 cells in vitro varied considerably between different strains.High attachment was observed with the chicken intestinal tract isolate Lactobacillus reuteri JCM1081（495.07±80.03）while low attachment showed in Lactobacillus acidphilus 1.1878（135.43±13.93）and Bifidobacterium longum B050102-14（47.17±5.84）.Lactobacillus reuteri JCM1081 was appeared on the intestinal mucosa surface diffusely.2.High adhesiveness strain Lactobacillus reuteri JCM1081 inhibit enteropathogenic Escherichia coli adhere to enterocyte-like cells and also reduce the invasiveness and enhance the integrity of the cells.The spent culture supernatant of the Lactobacillus acidphilus 1.1878 produces an antibacterial activity against enteropathogenic Escherichia coli.In vitro,no significant changes of the morphology,structure and function of the HT-29 cells were found after being treated with Lactobacilli for 3 hours,especially cellular LDH level,activity and permeability.Lactobacillus reuteri JCM1081 did not alter cell integrity and prevented the increase in permeability induced by enteropathogenic Escherichia coli infection.The distribution of F-actin was also not altered.In contrary,after HT-29 cells being infected by enteropathogenic Escherichia coli in vitro for 3 hours,the cellular activity decreased,the permeability of single layer cell increased and the distribution of F-actin were sparse or dot dome.The group of co-incubation showed that the degree of cell injury was less than the group of enteropathogenic Escherchia coli infection.The results showed that after de-contaminated and EPEC infected,Enterobacteria increased significantly,but anaerobic bacterial such as Bifidobacteria and Lactobacilli decreased and the ratio of anaerobes to aerobes declined,which lead to dysbiosis.The degree of dysbiosis in Lactobacillus feeding group was much less than that in non-feeding group,as well as the rate of organ bacteria count.3.Adherence of Lactobacillus reuteri JCM1081 and Lactobacillus acidphilus 1.1878 cells were significantly reduced by treatment with trypsin and protease.Which indicated that the adhesion of Lactobacillus to HT-29 cells appeared to be mediated by a cell surface component,and the cell surface determinant of the strain appeared to be proteinaceous. Extraction of the cell surface proteins with different solutions,were analyzed on SDS-PAGE and Western blotting with HRP-mucin and NHS-biotin labeled HT-29 cells. The strongest-reacting band that bound mucin and HT-29 cells yielded 14kD and 29kD subunit.The proteins which extracted from cell surface of Lactobacillus reuteri JCM1081 were performed to adhere to HT-29 cells,and elutes were analyzed on SDS-PAGE and Western blotting with anti-JCM1081 polyclonal antibodies.There have only one strongest-reacting band which yielded 29kD submit.The 29kD protein was purified and analyzed by mass spectrography,the results indicated that high degree similarities between 29kD protein from Lactobacillus reuteri JCM1081 and the protein lr0793 of Lacobacillus reuteri ATCC55730 strain（approx 71.1%identity） were confirmed.Which belong to the putative ATP-Binding Cassette transportor proteins superfamily. A highly significant loss of adhesion was observed when the spent culture supernatant of Lactobacillus reuteri JCM1081 was discarded or when it was replaced by fresh MRS culture medium.These results strongly suggest that a secretory component in the spent culture supernatant was involved in adhesion of Lactobacillus reuteri JCM1081.Treatment of the spent culture supernatant with trypsin or protease almost totally abolished the adhesiveness of Lactobacillus reuteri JCM1081.The SCS of Lactobacillus reuteri JCM1081 has no adhesion promoting effect on Lactobacillus acidphilus 1.1878.Extraction of promoting adhesion protein in spent culture supernatant of Lactobacillus reuteri JCM1081 with ammonium sulfate precipitation.These fractions were analyzed by SDS-PAGE and Western blotting with HRP-mucin.The reacting 29kD protein was found in them.Conclusion:1.An optimized Gram-stained method could be regarded as effective method to evaluate the adhesive ability of probiotics in vitro.2.The ability of the probiotics to adhere to the HT-29 cells in vitro varied considerably between strains and genus.Lactobacillus reuteri JCM1081 have a good ability to adhere to HT-29 cells and could colonize in intestinal mucosa of mice.3.Lactobacillus reuteri JCM1081 could protect HT-29 cells by its adhesive abilities which relates to its effectiveness.Lactobacillus reuteri JCM1081 interact with intestinal epithelial cell receptor to Competitively inhibit the adhesion and invasion of enteropathogenic Escherichia coli to HT-29 cells by incubation.Lactobacillus reuteri JCM1081 have ability to adhere and colonize to intestinal mucosa and form biologic barrier to enhance host resistance against enteropathogenic Escherichia coli infection.4.The adhesion of Lactobacillus reuteri JCM1081 to HT-29 cells appeared to be mediated by two components:a cell surface component and extracellular factor.The cell surface determinant of Lactobacillus reuteri JCM1081 appeared to be proteinaceous,and relative molecular mass which are 14kD and 29kD might be involved in adhesion of Lactobacillus to HT-29 cells.29kD protein could be adhesin of Lactobacillus reuteri JCM1081,which have high degree similarities with the protein lr0793 of Lacobacillus reuteri ATCC55730 strain（approx 71.1%identity） and belong to the putative ATP-Binding Cassette transportor proteins superfamily.A secretory proteinaceous component in the spent culture supernatant with relative molecular mass 29kD was considered to involve in adhesion of the Lactobacillus reutri JCM1081 too.The adhesion of strain JCM1081 was mediated by 29kD protein of bacteria secreted in the spent culture supernatant.更多还原