Impaired inactivation of digestive proteases:The possible key factor for the high susceptibility of germ-free and antibiotic-treated animals to gut epithelial injury

Recent study shows that germ-free and antibiotic-treated animals are highly susceptible to gut epithelial injury. This paper addresses that impaired inactivation of digestive proteases may be the key factor for the increased susceptibility.

The metabolism of constituents of Huangqin- Tang decoction,a traditional Chinese medicinal preparation,in conventional, germ-free and gnotobiote mice

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Colonization of germ-free mice with a mixture of three lactobacillus strains enhances the integrity of gut mucosa and ameliorates allergic sensitization

Increasing numbers of clinical trials and animal experiments have shown that probiotic bacteria are promising tools for allergy prevention. Here, we analyzed the immunomodulatory properties of three selected lactobacillus strains and the impact of their mixture on allergic sensitization to Bet v I using a gnotobiotic mouse model. We showed that Lactobacillus （L.） rhamnosus LOCK0900, L. rhamnosus LOCK0908 and L. casei LOCK0919 are recognized via Toll-like receptor 2 （TLR2） and nucleotide-binding oligomerization domain-containing protein 2 （NOD2） receptors and stimulate bone marrow-derived dendritic cells to produce cytokines in species- and strain-dependent manners. Colonization of germ-free （GF） mice with a mixture of all three strains （Lmix） improved the intestinal barrier by strengthening the apical junctional complexes of enterocytes and restoring the structures of microfilaments extending into the terminal web. Mice colonized with Lmix and sensitized to the Bet v I allergen showed significantly lower levels of allergen-specific IgE, IgG 1 and IgG2a and an elevated total IgA level in the sera and intestinal lavages as well as an increased transforming growth factor （TGF）-β level compared with the sensitized GF mice. Splenocytes and mesenteric lymph node cells from the Lmix-colonized mice showed the significant upregulation of TGF-β after in vitro stimulation with Bet v 1. Our results show that Lmix colonization improved the gut epithelial barrier and reduced allergic sensitization to Bet v 1. Furthermore, these findings were accompanied by the increased production of circulating and secretory IgA and the regulatory cytokine TGF-β. Thus, this mixture of three lactobacillus strains shows potential for use in the prevention of increased gut permeability and the onset of allergies in humans,

Establishment of an efficient germ-free animal system to support functional microbiome research

Germ-free animals are indispensable models for human and animal functional microbiome research Germ-free animals are animals that typically have no microorganisms living in or on them. These animals have become irreplaceable research tools for studying the relationships among single bacteria strains, multiple bacteria strains and hosts.

Gut microbiota absence and transplantation affect growth and intestinal functions:An investigation in a germ-free pig model

This study was conducted to investigate host-microbiota interactions and explore the effects of maternal gut microbiota transplantation on the growth and intestinal functions of newborns in a germ-free(GF)pig model.Twelve hysterectomy-derived GF Bama piglets were reared in 6 sterile isolators.Among them,6 were considered as the GF group,and the other 6 were orally inoculated with healthy sow fecal suspension as fecal microbiota transplanted(FMT)group.Another 6 piglets from natural birth were regarded as the conventional(CV)group.The GF and FMT groups were hand-fed with Co60-y-irradiated sterile milk powder,while the CV group was reared by lactating Bama sows.All groups were fed for 21 days.Then,all piglets and then were switched to sterile feed for another 21 days.Results showed that the growth performance,nutrient digestibility,and concentrations of short-chain fatty acids in the GF group decreased(P<0.05).Meanwhile,the serum urea nitrogen concentration and digesta pH values in the GF group increased compared with those in the FMT and CV groups(P<0.05).Compared with the CV group,the GF group demonstrated upregulation in the mRNA expression levels of intestinal barrier function-related genes in the small intestine(P<0.05).In addition,the mRNA abundances of intestinal development and absorption-related genes in the small intestine and colon were higher in the GF group than in the CV and FMT groups(P<0.05).The FMT group exhibited greater growth performance,lipase activity,and nutrient digestibility(P<0.05),higher mRNA expression levels of intestinal development and barrier-related genes in the small intestine(P<0.05),and lower mRNA abundances of pro-inflammatory factor in the colon and jejunum(P<0.05)than the CV group.In conclusion,the absence of gut microbes impaired the growth and nutrient digestibility,and healthy sow gut microbiota transplantation increased the growth and nutrient digestibility and improved the intestinal development and barrier function of newborn piglets,indicating the importance of intestinal microbes for intestinal development and functions.

Investigation of the metabolites of five major constituents from Berberis amurensis in normal and pseudo germ-free rats

Berberis amurensis(Berberidaceae)is a traditional Chinese medicine,which is often used to treat hypertension,inflammation,dysentery and enteritis.It contains alkaloids,mainly including berberine,berbamine,magnoflorine,jatrorrhizine and palmatine.Berberis amurensis extracts(BAEs)is often orally taken.Oral herbs might be metabolized by intestinal bacteria in the small intestine.However,the interaction between the herb and the gut microbiota is still unknown.In the current study,UPLC/Q-TOFMS/MS combined with Metabolitepilot and Peakview software was used to identify the metabolites of BAEs in anti-biotic cocktail induced pseudo germ-free rats and normal rats.As a result,a total of 46 metabolites in normal rats were detected and its main metabolic pathways include demethylation,dehydrogenation,methylation,hydroxylation,sulfation and glucuronidation.Only 29 metabolites existed in pseudo germ-free rats.Dehydrogenated metabolites(M29,M30,M34 and M36),methylated metabolites(M33,M41 and M46)and other metabolites were not detected in pseudo germ-free rats.The result implied that the intestinal bacteria have an influence on the metabolism of BAEs.Furthermore,this investigation might contribute to the understanding of the metabolism of BAEs,and further promote its clinical application.

Carrageenan oligosaccharides and associated carrageenan-degrading bacteria induce intestinal inflammation in germ-free mice

Carrageenans(CGNs)are widely used in foods and pharmaceuticals although their safety remains controversial.To investigate the effects of CGNs and CGN-degrading bacteria in the human colon,we screened for CGN degradation by human fecal microbiota,and for inflammatory response to CGNs and/or CGN-degrading bacteria in germ free mice.Thin-layer chromatography indicated that high molecular weight(MW)CGNs(!100 kDa)remained undegraded in the presence of human fecal microbiota,whereas low MW CGNs,i.e.,k-carrageenan oligosaccharides(KCO,~4.5 kDa)were degraded when exposed to seven of eight human fecal samples,although sulfate groups were not removed during degradation.Bacteroides xylanisolvens and Escherichia coli isolates from fecal samples apparently degraded KCO synergistically,with B.xylanisolvens serving as the primary degrader.Combined treatment of KCO with KCO-degrading bacteria led to greater pro-inflammatory effects in the colon and rectum of germ-free mice than either KCO or bacteria alone.Similarly,p-p38-,CD3-,and CD79a-positive immune cells were more abundant in combined treatment group mice than in either single treatment group.Our study shows that KCO-degrading bacteria and the low MW products of KCO can promote proinflammatory effects in mice,and represent two key markers for evaluating CGN safety in foods or medicines.

Characterizing the influence of gut microbiota on host tryptophan metabolism with germ-free pigs

Intestinal microbes are closely associated with host health,depending on metabolic crosstalk between the microbiota and host.Tryptophan metabolism is one of the best examples of metabolic crosstalk between intestinal microbiota and host;however,our understanding about the influence of intestinal microbiota on host tryptophan metabolism is limited.Thus,we established germ-free(GF)pig models to systemically explore the influence of intestinal microbiota on tryptophan metabolism.Five GF pigs were kept in GF conditions throughout the experiment(GF group).Six GF pigs were transplanted with fecal microbiota from donor sows to act as control pigs.Compared with control pigs,the GF pigs had remarkable alterations in tryptophan metabolism.The differential metabolites(P<0.05)were mainly found in the liver,circulation system and large intestine.Notably,the alteration of metabolites in tryptophan metabolism varied among organs,especially for the serotonin pathway.In GF pigs,tryptophan and kynurenine in the large intestine and 5-hydroxytryptophan in most organs were increased(P<0.05),while metabolites in the indole pathway in most organs were decreased(P<0.05).Collectively,our study reveals changes in tryptophan metabolism in GF pigs,highlighting the critical role of gut microbes in shaping host tryptophan metabolism.

Consistent Alterations of Human Fecal Microbes After Transplantation into Germ-free Mice

Fecal microbiota transplantation(FMT)of human fecal samples into germ-free(GF)mice is useful for establishing causal relationships between the gut microbiota and human phenotypes.However,due to the intrinsic differences between human and mouse intestines and the different diets of the two organisms,it may not be possible to replicate human phenotypes in mice through FMT;similarly,treatments that are effective in mouse models may not be effective in humans.In this study,we aimed to identify human gut microbes that undergo significant and consistent changes(i.e.,in relative abundances)after transplantation into GF mice in multiple experimental settings.We collected 16S rDNA-seq data from four published studies and analyzed the gut microbiota profiles from 1713 human–mouse pairs.Strikingly,on average,we found that only 47%of the human gut microbes could be re-established in mice at the species level,among which more than 1/3 underwent significant changes(referred to as“variable taxa”).Most of the human gut microbes that underwent significant changes were consistent across multiple human–mouse pairs and experimental settings.Consequently,about 1/3 of human samples changed their enterotypes,i.e.,significant changes in their leading species after FMT.Mice fed with a controlled diet showed a lower enterotype change rate(23.5%)than those fed with a noncontrolled diet(49.0%),suggesting a possible solution for rescue.Most of the variable taxa have been reported to be implicated in human diseases,with some recognized as the causative species.Our results highlight the challenges of using a mouse model to replicate human gut microbiota-associated phenotypes,provide useful information for researchers using mice in gut microbiota studies,and call for additional validations after FMT.An online database named FMT-DB is publicly available at http://fmt2mice.humangut.info/#/.

Evaluation of the Mechanisms Underlying Amino Acid and Microbiota Interactions in Intestinal Infections Using Germ-Free Animals

Intestinal infectious diseases refer to the inflammatory changes in the intestinal tract caused by pathogens(including bacteria,viruses,fungi,protozoa,or parasites)or their toxic products.A large number of microorganisms colonize the intestinal tract of healthy people,which together with the intestinal epithelium constitute the biological barrier of the intestinal tract to resist infectious diseases.As an“invisible organ,”the intestinal flora is closely related to human nutrition metabolism and intestinal infections.A variety of intestinal flora participates in the nutritional metabolism of amino acids,and the small molecular substances produced by the amino acid metabolism through the intestinal flora can enhance intestinal immunity and resist bacterial infections.In turn,amino acids can also regulate the composition of the intestinal flora,maintain the steady-state of the intestinal flora,protect the intestinal barrier,and inhibit colonization by pathogenic bacteria.As a model animal with a clear microbial background,germ-free(GF)animals can clarify the mechanisms of interactions between intestinal microbes and amino acid metabolism in intestinal infections by combining genetic engineering technology and multi-omics studies.This article reviews related researches on the involvement of intestinal microbes in host amino acid metabolism and resistance to intestinal infections and discusses the advantages of GF animal models for studying the underlying mechanisms.The GF animal model is helpful to further study the intervention effects of amino acid metabolism of targeted intestinal flora on intestinal infections.

共生微生物对仔猪肠道发育、代谢和线粒体功能的影响

【目的】以猪为动物模型研究共生微生物对宿主肠道发育代谢的调控作用。【方法】经无菌剖腹产、无菌饲养等技术培育无菌(germ-free,GF)仔猪和无特定病原(specific pathogen-free,SPF)仔猪,通过形态学观察、液相色谱分析、RNA-seq等方法研究共生微生物对仔猪肠道形态、代谢、基因表达及线粒体功能的影响。【结果】共生微生物对仔猪肠道的形态结构、短链脂肪酸含量、氨基酸代谢和肠道细胞线粒体含量等方面产生不同程度的影响;共生微生物影响仔猪肝脏、回肠和结肠组织的整体基因表达,调控线粒体氧化磷酸化、氨基酸代谢、脂质代谢等生物学过程相关基因表达,导致线粒体功能发生改变,从而影响仔猪肠道营养物质的吸收与代谢。【结论】共生微生物通过调控肠道细胞线粒体从而影响仔猪肠道发育和代谢,为“微生物-宿主互作”调控仔猪肠道健康相关研究奠定理论基础。

无菌鼠细菌检测方法的选择与探讨

目的 比较培养法、革兰染色镜检法和16S rRNA PCR方法对无菌小鼠粪便样品细菌检测的检测性能,为无菌鼠细菌检测方法的选择提供依据。方法 利用培养法、革兰染色镜检法、16S rRNA PCR方法,分别检测GF小鼠粪便、SPF小鼠粪便以及已知含菌量的测试样品(无菌小鼠粪便与金黄色葡萄球菌、大肠埃希菌混合,人为制作而成),验证3种方法对无菌小鼠粪便中细菌的检测限度;并同时使用3种方法检测临床样品,对比3种检测方法的优缺点,分析3种方法在实际检测中的可行性。结果 培养法、革兰染色镜检法和16S rRNA PCR方法检测灵敏度分别为102CFU/g、108CFU/g和106CFU/g,临床样品检测中阳性率均为1.36%(6/441)。结论 培养法、革兰染色镜检法、16S rRNA PCR法3种检测方法各有优缺点,需要联合使用,取长补短,确保检测结果的可靠。

无菌动物在肠道微生物组研究中的应用与发展

近年来,随着宏基因组概念的提出,人类宏基因组学也成为了揭示人体发育与健康的新途径。肠道微生物组作为人体内最大的微生物区系,已经被发现在人体正常生理功能和疾病的发生发展中具有重要的作用。肠道菌群有着丰富的种类及数量,在有关于肠道微生物组的研究中,无菌动物的应用使实验更具准确性,也更加便利,是一种比较理想的基础动物模型,不仅如此,在此基础上发展的模型也对该类研究有着重要意义。本综述就对无菌动物的认识、无菌动物在实验研究中的应用以及无菌动物模型的前景等方面,回顾无菌动物在肠道微生物组中的应用与发展。

几种活菌的定植性研究

由无菌动物和普通动物的实验证明，喂服双歧杆菌、乳杆菌及肠球菌可以在体内定植，双歧杆菌在构成膜菌群中起重要作用，其定植数为１０８～１０８ＣＦＵ／ｍｌ，在盲肠内容物中其定植数为１０８～１０９ＣＦＵ／ｍｌ，乳杆菌的定植数为１０８ＣＦＵ／ｍｌ，肠球菌的定植数为１０６～１０７ＣＲＦＵ／ｍｌ。

4种处理方法对月季种子萌发的影响

【目的】分析不同处理方法对月季种子萌发的影响,为有效提高月季种子发芽率及育种效率提供理论和技术支持。【方法】以月季种子为材料,开展低温层积处理、不同激素及浓度、不同播种基质及无菌播种对其萌发影响的研究。【结果】低温层积处理的月季种子发芽率为42.22%,发芽势为24.44%;100.0mg/LGA_3和100.0mg/LNAA处理可显著提高月季种子萌发率和发芽势(P<0.05),发芽率分别为55.56%和48.89%,发芽势分别为42.22%和30.00%;月季种子在河沙中发芽率为42.22%,发芽势为24.44%,优于泥炭:珍珠岩(体积比为2:1)及菜园土基质;在MS培养基中添加GA_3更有利于月季种子萌发,当GA_3浓度为0.5 mg/L时,其发芽率为55.56%,发芽势为34.44%。【结论】低温层积处理、100.0 mg/LGA_3和100.0 mg/LNAA浸泡月季种子可有效提高其萌发率;适宜的播种基质及培养基对月季种子的萌发有促进作用。

肿瘤坏死因子致伤无菌大鼠多器官功能损害的初步研究

本研究应用重组人的肿瘤坏死因子a型(rh-TNF-a)腹腔注射致伤无菌大鼠观察其症状。致伤72h后处死,观察血液、胸水、腹水肿瘤坏死因子(TNF)水平,脑、肝组织膜磷脂酶A_2及β-受体变化,对肺、肝、回肠巯基的影响以及部分致伤无菌大鼠腹腔液培养上清白细胞介素-1(IL-1)的水平。结果致伤组动物症状酷似实验动物的内毒素血症;大体解剖肝、脾、肺、脑、肠均有明显变化;膜磷脂酶A_2及β-受体均明显上升。肝、肺、回肠组织中巯基下降;IL-1水平上升。血液胸水、腹水TNF水平均显著上升。表明TNF对机体影响甚大,可影响多个脏器。

无菌动物的生物学特性及其在人类疾病研究中的应用

微生物学已开始从致病性研究为主转向了生理功能研究为主。正常菌群可促进机体代谢与肠道免疫系统的发育和成熟,也具有保护宿主对抗病原微生物入侵的作用。而研究肠道菌群的生理病理功能对动物模型提出了更高的要求。无菌(germ free,GF)动物的微生物背景清晰,且在解剖与形态学、代谢生理及免疫系统方面与常规实验动物相比有明显的特点,现已成为研究肠道菌群的生理病理功能的最佳动物模型。本文就GF动物在肠道微生态、免疫系统发育、代谢性疾病、药物代谢及肿瘤学等方面的研究进展作一综述。

无菌豚鼠与普通豚鼠血液学参数的比较

目的对普通级豚鼠进行剖宫产,培育无菌豚鼠模型,并比较无菌豚鼠和普通级豚鼠的血液学参数。方法行子宫摘除术摘除子宫,在无菌隔离器中将子宫剥离取仔,用人工哺乳的方法将仔豚鼠培育成无菌豚鼠,并在固定的周期检测豚鼠的无菌情况。采用全血细胞计数分析仪、全自动血液生化分析仪测定无菌豚鼠、普通豚鼠的血常规和血生化参数。用SPSS12.0进行统计分析。结果无菌豚鼠和普通豚鼠在白细胞(WBC)、嗜碱性粒细胞数(BA#)、单核细胞百分比(MO)、血红蛋白浓度(MCHC)、中性粒细胞数(NE#)、嗜酸性粒细胞数(EO#)、淋巴细胞数(LY#)七个血常规指标上有差异显著性,其他指标没有显著性差异;谷丙转氨酶(ALT)、乳酸脱氢酶(LDH)、总蛋白(TP)、白蛋白(ALB)、葡萄糖(GLU)、总胆固醇(CHO)、总胆红素(TBIL)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、甘油三酯(TG)、球蛋白(GLO)、A/G、K、Na、谷酰转肽酶(GGT)15个指标上差异有显著性,其他指标差异无显著性。结论通过规范的子宫摘除术和人工喂养的方法获得符合标准的无菌豚鼠,与普通豚鼠在多项血液学指标上差异有显著性。

思茅松成熟胚无菌萌发及农杆菌介导的遗传转化

以思茅松成熟胚为外植体,研究其适宜的萌发培养基,并对其根癌农杆菌介导的遗传转化进行初步研究。结果表明:适宜成熟胚萌发的培养基为1/2MS培养基;卡那霉素的质量浓度为40mg·L-1,抑菌剂羧苄青霉素的质量浓度为800mg·L-1;遗传转化的适宜条件为:菌液比色浓度的OD600为0.8,浸染时间15～30min,共培养时间1～2d;组织化学染色检测到了GUS瞬时表达。

挤压膨化对小麦胚芽储藏稳定性的影响

采用不同的挤压膨化工艺条件 ,研究其对小麦胚芽储藏稳定性的影响。结果表明 :小麦胚芽经挤压膨化后 ,可明显提高其储藏稳定性 ;当原料水分为 1 2 %、挤压机螺杆转速为 5 0 0 r·min- 1时 ,挤压膨化的小麦胚芽经 1 2 d烘箱快速储藏 ,其储藏稳定性表现最佳 ,脂肪酸值仅为 1 .0 6g· kg- 1 。