PCR-DGGE技术评价抗生素诱导的肠道菌群失调

目的 利用PCR-DGGE技术结合活菌计数评价抗生素引起的肠道菌群失调.方法 SPF级BALB/c雌性小鼠连续4 d灌胃头孢曲松溶液125 mg/ml,0.4 ml/d,运用基于细菌16S DNA的PCR-DGGE技术结合活菌计数分析小鼠肠道菌群变化.结果 活菌计数结果与PCR-DGGE图谱显示,头孢曲松处理3 d后小鼠肠道菌群出现严重失调.结论 PCR-DGGE技术可直观而灵敏地显示抗生素处理过程中肠道菌群的动态变化,适用于评价抗生素引起的肠道菌群失调.     

加替沙星对小鼠肠道菌群的影响

目的 了解加替沙星正常剂量用药和非正常剂量用药方式对小鼠肠道菌群的影响.方法 24只昆明小鼠随机分为3组,每组8只,对照组只灌胃生理盐水,另外2组分别按正常剂量和正常剂量的一半给小鼠灌胃加替沙星溶液,7 d后无菌采取小鼠粪便,观察各组小鼠粪便中双歧杆菌、类杆菌、乳酸杆菌、大肠埃希菌及肠球菌数量.结果 加替沙星灌胃7 d后,与给药前相比,肠杆菌和肠球菌药敏结果无明显变化.灌胃前后肠杆菌对CIP、LEV、GAT、AM、CRO、AMK、NOR均敏感,肠球菌对AM、LEV、VA、GAT均敏感,对NOR、CIP、CRO敏感性不同.2种用药方式可导致大肠埃希菌数量显著减少(P＜0.05),肠球菌数量稍有增加(P＞0.05).而双歧杆菌、乳酸杆菌和类杆菌数量无明显变化(P＞0.05).结论 加替沙星2种用药方式短时期对肠道菌群影响不大(肠杆菌除外),不易产生耐药性.其杀灭肠杆菌的作用远大于对厌氧菌的作用.     

残留剂量恩诺沙星对SPF小鼠肠道菌群数量和多样性的影响研究

目的研究不同残留剂量的恩诺沙星对SPF小鼠肠道菌群数量和种类的影响。方法30只SPF雌性KM小鼠,随机分为5组。根据中国现行规定恩诺沙星的日允许摄入量（ADI）设置剂量（0mg／L、0．03mg／L、0．3mg／L、3mg／L、30mg／L）,0．3mg／L相当于ADI。适应性饲养7d后给药35d,恢复21d。使用活菌计数法检测模型小鼠粪便菌群数量的变化。使用PCR—DGGE法检测粪便菌群多样性的变化。结果3mg／L和30mg／L组总需氧菌的数量与对照组比较差异有显著性（P〈0．05或P〈0．01）;30mg／L组的肠杆菌的数量和对照组比较差异有非常显著性（P〈0．01）,其余种类细菌数量差异没有显著性。PCR．DGGE结果显示0．3mg／L的恩诺沙星对小鼠肠道菌群多样性产生影响,3mg／L和30mg／L两组影响显著。结论PCR—DGGE技术是一种比活菌计数更灵敏的实验方法,可以用于抗生素残留微生物安全性评价;中国现行规定恩诺沙星的日允许摄入量（ADI）可能会对人体肠道菌群产生影响。     

玉米紫色植株花色苷色素对小鼠肠道菌群的影响

目的了解玉米紫色植株花色苷色素对小鼠肠道菌群的影响。方法按中华人民共和国卫生部《保健食品检验与评价技术规范》中的动物实验调节肠道菌群功能的检验方法,观察玉米紫色植株色素对小鼠肠道中肠杆菌、乳杆菌、双歧杆菌数量的影响。结果灌服后小鼠肠道乳杆菌、双歧杆菌数量变化差异均无显著性,低剂量组肠扦菌数量明显低于灌服前（P〈0．05）,其他组肠杆菌数量变化差异无显著性。结论玉米紫色植株色素对小鼠肠道菌群可能有影响。     

合生元益生菌冲剂对小鼠肠道菌群调节作用的研究

目的 研究合生无益生菌冲剂对小鼠肠道菌群的调节作用.方法 将小鼠分为阴性对照组、合生元益生菌冲剂组.阴性对照组灌服蒸馏水14 d,合生元益生菌冲剂组灌服1 g/kg·d剂量的台生元益生菌冲剂14 d,检测实验前后肠道菌群数量.结果 灌服后小鼠肠道菌群与灌服前比较,,双歧杆菌数量有显著性增加（P＜0.05）,肠杆菌、肠球菌、乳杆菌和产气荚膜梭菌数量有无显著性变化（P＞0.05）.结论 合生元益生菌冲剂对小鼠肠道菌群具有一定的调节作用.     

老年房颤患者肠道菌群变化及其影响因素

目的探讨老年房颤患者肠道菌群变化及影响菌群变化的相关因素。方法回顾性收集20例老年房颤患者作为房颤组,并选取性别、年龄与患者匹配的30例老年健康体检者作为对照组,分析两组对象肠道优势菌的特点。同时收集房颤组患者C反应蛋白(CRP)、总胆汁酸(TBA)、同型半胱氨酸(Hcy)、B型纳尿肽(BNP)、左室射血分数数值(LVEF)等检测结果,分析上述因素与肠道优势菌群的关系。结果房颤组患者直肠真杆菌数量为(4.66±1.62)lg copies/g粪便,低于对照组的(6.10±1.44)lg copies/g粪便,差异有统计学意义(t=-3.286,P=0.002)。房颤组患者肠道普拉梭菌、肠球菌、类杆菌、乳杆菌、双歧杆菌、柔嫩梭菌、酪酸梭菌、Atopobium cluster(Ato)、肠杆菌数量及双歧杆菌/肠杆菌(B/E)值与对照组比较差异无统计学意义(均P0.05)。在房颤组中,CRP升高患者肠道双歧杆菌数量和B/E值显著高于CRP正常患者,CRP升高与双歧杆菌数量呈正相关(r=0.651,P=0.002);Hcy升高患者肠道肠杆菌数量显著高于Hcy正常患者,Hcy升高与肠杆菌数量呈正相关(r=0.567,P=0.009)。BNP、TBA、LVEF与优势菌群无显著相关性。结论老年房颤患者存在肠道菌群失调。CRP、Hcy和老年房颤患者肠道菌群具有一定的相关性。     

双叉奶中合生元对改善肠道菌群功效的研究

目的 对双叉奶中合生元改善肠菌群的功效进行研究分析。方法 依卫生部《新增保健功能评价指标及程序的初步方案》,做动物及人体改善肠道菌群试验。结果 1．双叉奶中合生元对小鼠4种肠道菌群数量的影响：与对照组比较,各剂量组小鼠肠道内肠球菌、肠杆菌数量均无显著性差异（P＞0．05）;低剂量、高剂量组乳酸杆菌数量明显增加,有显著性差异（P＜0．05）,中剂量组乳酸杆菌数量增加显著,有非常显著性差异（P＞0．01）;低剂量、中剂量组双歧杆菌数量明显增加,有显著性差异（P＞0．05）。2．双叉奶中合生元对人体5种肠道菌群数量的影响：与饮用前相比,受试人群肠道内肠杆菌、肠球菌数量均无显著性差异（P＞0．05）;双歧杆菌、乳酸杆菌数量增加显著,有非常显著性差异（P＞0．01）。肠梭菌数量降低显著,有非常显著性差异（P＞0．01）。3．受试人群自觉症状观察：所有人无不良反应,可改善便秘、增加食欲、排气。结论 双叉能奶中合生元对小鼠、人体肠道菌群具改善作用。     

不同剂量长双歧杆菌对抗生素诱导大鼠肠道菌群失调的疗效观察

目的观察不同剂量长双歧杆菌对抗生素诱导大鼠腹泻及肠道菌群失调的调节作用。方法按体表面积予头孢曲松钠钠腹腔注射诱导大鼠腹泻及肠道菌群失调,然后灌胃不同剂量的长双歧杆菌菌粉,观察治疗后大鼠肠道菌群数量、B/E值和血清内毒素的变化。结果予头孢曲松钠钠处理后,大鼠肠道内双歧杆菌数量明显减少,肠球菌异常增殖,B/E值明显下降,血清内毒素升高,与对照组相比差异有统计学意义(P0.05)。高倍剂量治疗组肠道内细菌数量恢复程度及B/E值改善程度显著优于低倍剂量组。长双歧杆菌各治疗组的血清内毒素含量无明显改善,与自然恢复组之间无差异。结论头孢曲松钠可诱导大鼠肠道菌群失调。高倍剂量长双歧杆菌对头孢曲松钠所致肠道菌群失调效果更佳,短期应用不能明显改善头孢曲松钠诱发的内毒素血症。     

灭活双歧杆菌调整小鼠抗生素相关性菌群失调

目的：观察灭活的双歧杆菌及其耗尽培养上清液（SCS）对小鼠肠道生理菌群的影响。方法：应用腹腔注射青霉素造成肠菌群失调动物模型,分别以灭活的双歧杆菌菌液,耗尽培养上清液以及活菌菌液对菌群失调小鼠进行灌胃治疗。结果：活菌组、死菌组及SCS组同自然恢复组的肠道生理菌群相比差异均有显著性,死菌组与SCS组相比,差异也有显著性。结论：灭活的双歧杆菌及其SCS对小鼠肠道菌群失调的恢复具有调整作用,尤其对双歧杆菌和乳酸杆菌有更明显的扶持作用。     

肝硬化患者口服培菲康和妈咪爱对肠道菌群影响的研究

目的:研究肝硬化患者肠道菌群的特点,观察患者口服妈咪爱和培菲康前后肠道菌群、粪便pH及血氮水平的变化.方法:选择肠道菌群中具有代表性的细菌共4种进行培养和计数.选择20例正常人为对照组,计数肠道菌群中4种细菌的数量.40例肝硬化患者随机分成2组,分别给予培菲康(三联活菌)及妈咪爱(二联活菌)共治疗14 d.观察治疗前后肠道菌群落计数、粪便pH、血氨水平.结果:①肝硬化组患者存在不同程度的肠道菌群失调,主要表现为双歧杆菌减少(P＜0.05),而肠球菌、肠杆菌则显著增多(P＜0.01)②菌群失调的严重程度随患者肝功能严重程度而加重.③临床表现有腹泻的肝硬化患者,肠道总体菌量和厌氧菌数量均低于非腹泻患者,以双歧杆菌(P＜0.01)减少为著.④两种益生菌制剂均可提高肝硬化患者肠道双歧杆菌的数量(P＜0.05),并可降低血氨和粪便pH(P＜0.05).结论:①肝硬化组患者存在肠道菌群失调.②益生菌制剂均可有效改善肝硬化患者肠道菌群失调并可降低血氨和粪便pH.     

小鼠肠道菌群失衡模型建立

目的利用抗生素干扰小鼠肠道菌群建立轻重程度的菌群失衡小鼠模型。方法选用不同浓度的头孢曲松钠,行小鼠灌胃,取盲肠内容物连续观察培养优势菌群变化。结果优势菌群失衡组小鼠与对照比较,盲肠体积增大,盲肠指数升高。头孢曲松钠8g/(kg·d)剂量连续灌胃8d,造成重度失衡模型,小鼠肠道只能检出肠杆菌属、链球菌属,且数量被抑制在103CFU/g以下。头孢曲松钠5g/(kg·d)剂量连续灌胃8d,造成轻度失衡模型,小鼠肠道双歧杆菌属、链球菌属和韦荣球菌属数量降至105左右,类杆菌属降至104左右。消化球菌属降至103左右,而其他菌属如乳酸杆菌属、肠球菌属和肠肝菌属等数量显著降低。结论亚致死剂量头孢曲松钠灌胃可以建立肠道菌群重度失衡模型。     

SPF级KM小鼠与BALB/c小鼠肠道总菌多样性的比较

目的分析SPF级封闭群KM小鼠及近交系BALB/c小鼠的肠道菌群总菌多样性,比较两个不同遗传背景肠道总菌的丰富度、Shannon-Wiener指数和均匀度。方法收集SPF级KM小鼠和BALB/c小鼠新鲜粪便,提取粪便总菌DNA,用基于细菌16S rDNA序列的变性梯度凝胶电泳(PCR-DGGE)分析粪便总菌多样性。结果 SPF级KM小鼠及BALB/c小鼠粪便总菌多样性差异无统计学意义(P0.05),品系内不同性别之间粪便总菌多样性差异亦无统计学意义(P0.05)。结论选择SPF级小鼠进行微生态学相关研究时,封闭群KM小鼠及近交系BALB/c小鼠均可作为选择对象,同时可忽略菌群的性别差异。     

益生元对抗生素引起的肠道菌群失调的作用

目的 评价益生元菊糖和大蒜多糖在治疗肠道菌群失调中的作用.方法 72只Balb/c小鼠随机分为6组,随机取出12只作为正常对照组,其余灌胃盐酸林可霉素和头孢曲松钠混合抗生素溶液3d,建立小鼠肠道失调模型后,以生理盐水灌胃作为自然恢复组,其余4组分别灌胃菊糖、低剂量大蒜多糖、高剂量大蒜多糖以及丽珠肠乐5d.受试小鼠无菌取粪,观察各组肠道菌群的数量变化、测量血清IgG,粪便内毒素含量、有机酸含量变化.结果 菊糖和大蒜多糖高剂量灌胃能加速恢复抗生素导致的小鼠肠道菌群失调,这两种益生元对内毒素的清理、免疫力的恢复也有积极的效果,效果均优于丽珠肠乐和自动恢复.结论 益生元有加快肠道菌群恢复和降低抗生素的破坏效果,有益于小鼠肠道健康.     

Systemic control of plasmacytoid dendritic cells by CD8+ T cells and commensal microbiota

The composition of the intestinal microbial community is a distinctive individual trait that may divergently influence host biology. Because dendritic cells (DC) regulate the quality of the host response to microbiota, we evaluated DC in mice bearing distinct enteric microbial communities divergent for colitis susceptibility. Surprisingly, a selective, systemic reduction of plasmacytoid dendritic cells (pDC) was observed in isogenic mice with different microbiota: restricted flora (RF) vs specific pathogen free (SPF). This reduction was not observed in germfree mice, suggesting that the pDC deficiency was not simply due to a lack of intestinal microbial products. The microbial action was linked to cytotoxic CD8(+) T cells, since pDC in RF mice were preserved in the CD8(-/-) and perforin(-/-) genotypes, partially restored by anti-CD8beta Ab, and augmented in SPF mice bearing the TAP(-/-) genotype. Direct evidence for pDC cytolysis was obtained by rapid and selective pDC depletion in SPF mice transferred with RF CD8(+) T cells. These data indicate that commensal microbiota, via CTL activation, functionally shape systemic immune regulation that may modify risk of inflammatory disease.     

Gram-negative bacteria aggravate murine small intestinal Th1-type immunopathology following oral infection with Toxoplasma gondii

Oral infection of susceptible mice with Toxoplasma gondii results in Th1-type immunopathology in the ileum. We investigated gut flora changes during ileitis and determined contributions of gut bacteria to intestinal inflammation. Analysis of the intestinal microflora revealed that ileitis was accompanied by increasing bacterial load, decreasing species diversity, and bacterial translocation. Gram-negative bacteria identified as Escherichia coli and Bacteroides/Prevotella spp. accumulated in inflamed ileum at high concentrations. Prophylactic or therapeutic administration of ciprofloxacin and/or metronidazole ameliorated ileal immunopathology and reduced intestinal NO and IFN-gamma levels. Most strikingly, gnotobiotic mice in which cultivable gut bacteria were removed by quintuple antibiotic treatment did not develop ileitis after Toxoplasma gondii infection. A reduction in total numbers of lymphocytes was observed in the lamina propria of specific pathogen-free (SPF), but not gnotobiotic, mice upon development of ileitis. Relative numbers of CD4(+) T cells did not differ in naive vs infected gnotobiotic or SPF mice, but infected SPF mice showed a significant increase in the frequencies of activated CD4(+) T cells compared with gnotobiotic mice. Furthermore, recolonization with total gut flora, E. coli, or Bacteroides/Prevotella spp., but not Lactobacillus johnsonii, induced immunopathology in gnotobiotic mice. Animals recolonized with E. coli and/or total gut flora, but not L. johnsonii, showed elevated ileal NO and/or IFN-gamma levels. In conclusion, Gram-negative bacteria, i.e., E. coli, aggravate pathogen-induced intestinal Th1-type immunopathology. Thus, pathogen-induced acute ileitis may prove useful to study bacteria-host interactions in small intestinal inflammation and to test novel therapies based on modulation of gut flora.     

Microbiota transplantation reveals beneficial impact of berberine on hepatotoxicity by improving gut homeostasis

Berberine has been shown to reduce acute liver injury although the underlying mechanism is not fully understood. Because of the anatomic connection, the liver is constantly exposed to gut-derived bacterial products and metabolites. In this study, we showed that berberine has beneficial effects on both hepatotoxicity and intestinal damage in a rat model of chronic or acute liver injury. Microbiota transplantation from the rats with chronic hepatotoxicity could aggravate acute hepatotoxicity in mice treated with diethylnitrosamine (DEN). In rat models with gut homeostasis disruption induced by penicillin or dextran sulfate sodium (DSS), their fecal microbiota could also cause an enhanced hepatotoxicity of recipient mice. When treated with berberine, the DSS-induced enteric dysbacteriosis could be mitigated and their fecal bacteria were able to reduce acute hepatotoxicity in recipient mice. This study indicates that berberine could improve intestinal dysbacteriosis, which reduces the hepatotoxicity caused by pathological or pharmacological intervention. Fecal microbiota transplantation might be a useful method to directly explore homeostatic alteration in gut microbiota.     

Impacts of ceftriaxone exposure during pregnancy on maternal gut and placental microbiota and its influence on maternal and offspring immunity in mice

This study aimed to investigate the association between microbiota found in the maternal gut and placenta, and whether ceftriaxone exposure during pregnancy could alter these microbiota, and consequently affect the immunity of the mothers and their offspring. The microbiota in the feces and placenta of the dams were comprehensively analyzed using16S rRNA sequencing. Furthermore, viable bacteria in the placentas and blood of pups were also isolated by plate cultivation then taxonomically identified in detail by clone sequencing. Serum cytokines collected from dams and pups were quantitatively profiled using Luminex. The spleen organ index of dams was significantly lower and the offspring serum interleukin-6 levels were significantly higher in ceftriaxone-treated mice compared with the control group. The maternal fecal microbiota community was drastically altered in ceftriaxone-treated mice with significantly decreased diversity, depletion of Bacteroidetes and the blooming of Tenericutes. However, the placenta microbiota was dominated by Proteobacteria especially characteristically by Ralstonia, which was distinct from the maternal gut microbiota, regardless of whether ceftriaxone treatment or not. Viable bacteria have been found in placenta and blood cultures. These results indicated that ceftriaxone exposure in pregnancy could dramatically alter maternal intestinal microbiota, which affected the immunity of the mothers and their offspring at least partly, characteristically by enhanced pro-inflammatory responses. This study also indicated that the placenta might harbor its own microbes and the microbes were distinct from maternal gut microbiota, which may not be affected by oral administration of ceftriaxone during pregnancy.     

荷瘤裸鼠暴露于PM2.5后肠道菌群的急性改变

目的研究暴露于PM2.5后的肺癌荷瘤裸鼠肠道菌群发生的急性改变。方法 18只Bac裸鼠随机分为PM2.5暴露组和对照组,每组9只。全部裸鼠应用A549细胞腋下注射后,饲养1周让其适应环境,第2周开始于动式染尘暴露仓中暴露染尘,对照组暴露于生理盐水。每天暴露2h,每周暴露6d,共暴露3周。利用16S rDNA分析技术对粪便标本的PCR产物进行高通量测序。结果暴露于PM2.5的肺癌荷瘤裸鼠肠道菌群结构发生改变,与对照组荷瘤裸鼠肠道菌群相比,其厚壁菌门、拟杆菌门细菌显著减少,而致病菌变形菌门细菌增加。结论 PM2.5可以导致肺癌荷瘤裸鼠肠道菌群失衡。