肠道微生态在华支睾吸虫致宿主肝功能损伤中的作用研究

目的研究肠道微生态在华支睾吸虫致肝损伤中的作用。方法建立华支睾吸虫感染大鼠模型。分别在造模后48 h、18 d和35 d检测血浆内毒素、ALT(谷丙转氨酶)、AST(谷草转氨酶)水平;检测肠黏液中sIgA含量;取肠内容物进行乳酸杆菌、双歧杆菌、肠球菌和肠杆菌的培养及定量分析。结果造模后18 d、35 d,大鼠血浆内毒素、ALT、AST水平明显升高,差异有统计学意义(t_内=8.335、7.612,t_(ALT)=11.815、9.874,t_(AST)=7.433、8.015,P值均0.01);造模后48 h,肠黏液sIgA水平高于正常组(t=2.752,P0.05),尤以造模后18 d、35 d升高显著(t=13.118、9.546,P值均0.01);造模后48 h、18 d和35 d,肠道乳酸杆菌和双歧杆菌数量明显减少(t_乳=2.612、4.142、5.556,t_(双)=2.302、4.565、3.982,P值0.05或P值0.01),造模后18 d和35 d,肠杆菌和肠球菌数量明显增多(t_肠=4.562、5.247,t肠=5.366、4.775,P值均0.01)。结论华支睾吸虫感染可致大鼠肠道菌群发生失调,引发内毒素血症,参与介导对肝细胞的损伤。     

参麦注射液对严重烫伤大鼠肠道屏障功能的保护作用

目的:探索参麦注射液对30% Ⅲ°烫伤早期肠道屏障功能的保护作用,为参麦注射液防治肠源性感染提供实验依据。方法:Wistar大鼠60只,随机分为正常对照组、模型对照组,地塞米松5 mg/kg组、参麦注射液5、10、15 mg/kg组,每组10只,使用烫伤仪建立30% Ⅲ°烫伤动物模型,立即腹腔注射相应的药物,每天1次。烫伤72 h后,检测肝脏、脾脏、肠系膜淋巴结细菌移位量、血浆内毒素、二胺氧化酶(DAO)、肿瘤坏死因子-α(TNF-α)及白细胞介素-6(IL-6)水平和肠粘膜分泌型免疫球蛋白A(sIgA)的水平。结果:与正常对照组比较,模型组肝脏、脾脏、肠系膜淋巴结细菌移位量,血浆内毒素、DAO、TNF-α及 IL-6和肠黏膜sIgA水平明显升高(P＜0.01);与模型组比较,地塞米松组和参麦注射液5、10、15 mg/kg组肝脏、脾脏、肠系膜淋巴结细菌移位量,血浆内毒素、DAO、TNF-α及 IL-6和肠黏膜sIgA水平明显降低(P＜0.05或P＜0.01)。结论:参麦注射液可减轻严重烫伤引起的肠粘膜损伤,效果与地塞米松相当,高剂量组效果更好。     

Liver Ischemic Preconditioning (IPC) Improves Intestinal Microbiota Following Liver Transplantation in Rats through 16s rDNA-Based Analysis of Microbial Structure Shift

Background

Ischemia-reperfusion (I/R) injury is associated with intestinal microbial dysbiosis. The “gut-liver axis” closely links gut function and liver function in health and disease. Ischemic preconditioning (IPC) has been proven to reduce I/R injury in the surgery. This study aims to explore the effect of IPC on intestinal microbiota and to analyze characteristics of microbial structure shift following liver transplantation (LT).

Methods

The LT animal models of liver and gut IPC were established. Hepatic graft function was assessed by histology and serum ALT/AST. Intestinal barrier function was evaluated by mucosal ultrastructure, serum endotoxin, bacterial translocation, fecal sIgA content and serum TNF-α. Intestinal bacterial populations were determined by quantitative PCR. Microbial composition was characterized by DGGE and specific bacterial species were determined by sequence analysis.

Principal Findings

Liver IPC improved hepatic graft function expressed as ameliorated graft structure and reduced ALT/AST levels. After administration of liver IPC, intestinal mucosal ultrastructure improved, serum endotoxin and bacterial translocation mildly decreased, fecal sIgA content increased, and serum TNF-α decreased. Moreover, liver IPC promoted microbial restorations mainly through restoring Bifidobacterium spp., Clostridium clusters XI and Clostridium cluster XIVab on bacterial genus level. DGGE profiles indicated that liver IPC increased microbial diversity and species richness, and cluster analysis demonstrated that microbial structures were similar and clustered together between the NC group and Liver-IPC group. Furthermore, the phylogenetic tree of band sequences showed key bacteria corresponding to 10 key band classes of microbial structure shift induced by liver IPC, most of which were assigned to Bacteroidetes phylum.

Conclusion

Liver IPC cannot only improve hepatic graft function and intestinal barrier function, but also promote restorations of intestinal microbiota following LT, which may further benefit hepatic graft by positive feedback of the “gut-liver axis”.     

异麦芽低聚糖对衰老模型小鼠肠黏膜免疫功能调节作用的研究

目的探讨异麦芽低聚糖（Isomalto oligosaccharide,IMO）对衰老模型小鼠肠黏膜免疫功能的调节作用及可能机制。方法昆明纯系小鼠随机分为Young组、Aging组、IMO组和IMOLCM组。采用D-半乳糖造成衰老模型后,给予相应药物干预。采用细菌定量测定法检测肠道菌群、放射免疫法检测肠黏膜sIgA、免疫组化法检测肠黏膜IgA^＋浆细胞的表达。结果与Young组相比,Aging组小鼠存在肠道菌群失调、肠黏膜sIgA含量降低、IgA^＋浆细胞表达减少（P〈0．05）;与Aging组相比,IMO组肠道菌群失调状况有所改善,肠黏膜sIgA含量增加、IgA^＋浆细胞的表达增加（P〈0．05）;与IMO组相比,IMOLCM组肠道菌群失调再次出现,肠黏膜sIgA降低、IgA^＋浆细胞的表达降低（P〈0．05）。结论异麦芽低聚糖可改善衰老模型小鼠肠道菌群失调状态和提高肠黏膜免疫功能;异麦芽低聚糖提高肠黏膜免疫功能可能主要由增加益生菌数量间接实现的。     

大承气汤对MODS时肠道细菌微生态学影响的实验研究

目的探讨多器官功能不全综合征(MODS)大鼠肠道细菌微生态的变化及其与肠源性内毒素血症和细菌易位的关系,并观察大承气汤的影响。方法32只SD大鼠随机分成4组,对照组、模型组、大承气组和氨苄青霉素组。腹腔注射无菌酵母多糖A制备大鼠MODS模型。各组动物于造模后48 h无菌操作抽取外周静脉血和门静脉血进行内毒素含量测定;取肠系膜淋巴结进行细菌定量培养,取回肠和盲肠内容物进行肠腔内游离内毒素测定;取盲肠内容物进行肠道细菌微生态学分析。结果模型组外周血和门静脉血内毒素水平以及肠腔内游离内毒素含量均明显高于对照组(P<0.05);与对照组相比,模型组肠道菌群出现明显变化。肠球菌、肠杆菌数量明显增加,而双歧杆菌和乳酸杆菌数量出现显著下降,类杆菌数量亦出现明显下降(P<0.05)。模型组厌氧菌总数明显下降而需氧菌总数明显增加,同时厌氧菌总数/需氧菌总数的比值和B/E比值呈相应下降,发生倒置(P<0.05);正常对照组未发现肠道细菌向肠系膜淋巴结的易位,而模型组细菌易位阳性率是83.33%(P<0.05)。与模型组相比,大承气汤组上述各指标均出现明显变化(P<0.05);抗生素组作用不明显(P>0.05)。结论MODS时大鼠肠道细菌微生态出现明显变化,发生肠源性内毒素血症和细菌易位。大承气汤可以调整肠道菌群,恢复肠道微生态平衡,增加机体定植抗力,防治细菌易位和内毒素血症。     

Intestinal microecology in some systemic connective tissue diseases

In patients with systemic connective tissue diseases were studied. The species structure intestinal microflora was characterized by decrease of dominating status of anaerobic bacteria and increase of role of opportunistic bacteria. The number of Lactobacillus decreased significantly. Bifidobacterium and Bacteroides was also decreased in number while their detection rate did not change. Opportunistic bacteria with hemolytic activity acquired greater significance in the forming of patients' intestinal microbiocenosis. Significant increase of Staphylococcus and opportunistic Enterobacteriaceae detection rate and density of intestine colonization was detected. Colonization of the intestine by S. aureus was revealed. Comparative analysis of the qualitative and quantitative structure of the intestinal microflora in patients with systemic lupus erythematosus and systemic scleriasis revealed similar patterns. However, colonization of the gut by opportunistic intestinal bacteria, Candida and microorganisms with hemolytic activity was more frequently observed in patients with systemic scleriasis.     

多器官功能不全综合征时肠道细菌微生态学改变的实验研究

为研究多器官功能不全综合征（ＭＯＤＳ）时肠道细菌微生态的变化以及Ｇ＾－杆菌消长与肠道和血液中内毒素水平的关系,本实验选用ＳＤ大鼠,应用无菌Ｚｙｍｏｓａｎ腹腔注射制备大鼠ＭＯＤＳ模型,并对ＭＯＤＳ大鼠肠道菌群进行定量分析,对门静脉和外周静脉血中的内毒以及肠道游离内毒素含量进行定量测定。结果发现,ＭＯＤＳ状态下肠道细菌微生态发生明显变化,表现为肠杆菌和肠球菌等肠道内需氧菌的数量明显增多,双歧杆菌和乳酸     

Intestinal microflora and concomitant gastrointestinal diseases in patients with chronic hepatitis B and C

In chronic viral hepatitis B and C the development of intestinal dysbacteriosis and high occurrence of concomitant diseases of the gastrointestinal tract were observed. In cases of increased dysbacteriosis degree and in the presence of concomitant diseases the blood plasma of patients exhibited higher activity in reaction with the of amebocytes lysate obtained from crabs of the genus Limulus. A suggestion was made that the endotoxin of Gram negative intestinal microflora could probably play some role in the development of pathological processes in chronic viral hepatitis B and C.     

肠道菌群及内毒素在多器官功能不全综合征时的变化

目的　探讨肠道菌群及内毒素在多器官功能不全综合征( MODS)时的变化。方法　取SD大鼠,腹腔注射无菌酵母多糖A制备MODS模型,检测大鼠肠道菌群、外周血和门静脉血中的内毒素以及肠道游离内毒素含量,并进行定量分析。结果　模型组大鼠肠道专性厌氧菌的数量明显减少,革兰阴性杆菌和双歧杆菌的比例倒置,内毒素含量明显增加,与对照组比差异有显著性( P<0 .0 5 )。结论　MODS时肠道细菌微生态发生明显改变,肠道内毒素池与肠道革兰阴性杆菌的变化密切相关     

肠道菌群变化对实验小鼠肠黏膜免疫的影响

目的探讨肠道菌群变化对肠黏膜相关淋巴组织的影响。方法通过变性梯度凝胶电泳（Denatu-ring gradient gel electrophoresis,DGGE）法研究了三种不同级别实验小鼠即清洁级小鼠、SPF小鼠和普通小鼠肠道菌群的组成,并用免疫组织化学（immunohistochemistry,IHC）方法研究了此三种不同级别的实验小鼠肠黏膜相关淋巴组织sIgA阳性细胞分布情况。结果普通小鼠肠道细菌种类最多,其sIgA阳性细胞分布最多,肠道不同部位之间sIgA分布情况差异有显著性（P〈0.05）,小肠和大肠之间的阳性细胞分布差异极显著（P〈0.01）;其次是清洁级小鼠,其肠道不同部位之间菌种组成差异无显著性,小肠和大肠之间的阳性细胞分布差异有显著性（P〈0.05）;SPF小鼠肠道细菌种类最少,故其sIgA阳性细胞分布最少,且其肠道不同部位之间菌种组成差异无显著性,小肠和大肠之间的阳性细胞分布差异无显著性（P〉0.05）。结论随着动物微生物控制级别的增高,肠道微生物多样性递减;sIgA阳性细胞与肠道细菌种类正相关。     

Does the intestinal microflora synthesize pyrroloquinoline quinone?

Pyrroloquinoline quinone (PQQ) functions as a cofactor for prokaryotic oxidoreductases, such as methanol dehydrogenase and glucose dehydrogenase. When chemically-defined diets without PQQ are fed to animals, lathyritic changes are observed. In previous studies, it was assumed that PQQ was produced by the intestinal microflora; consequently, antibiotics were routinely added to diets. In the present study this assumption is tested further in mice by: (i) examining the effects of dietary antibiotics on fecal PQQ excretion, (ii) isolating the intestinal flora to identify bacteria known to synthesize PQQ and (iii) determining in vitro if the intestinal microflora synthesizes PQQ from radio-chemically labeled precursors. The results of these experiments indicate that little if any PQQ is synthesized by the intestinal microflora. Rather, when PQQ is present in the intestine, the diet is a more obvious source.     

Intestinal Microflora Associated with Enteritis of Early-Weaned Pigs

Studies were conducted on the enumeration of 7 groups of fecal microflora including total aerobes, total anaerobes, coliforms, lactobacilli, staphylococci, streptococci and yeasts and molds of 18-day old piglets. These pigs were early weaned (21 days) on different modifications of an early-weaning ration. The above mentioned microflora were enumerated again when some of the pigs in a replicate started scouring. The occurrence of diarrhea was always associated with significant increases in the numbers of coliforms and corresponding decreases in the lactobacilli counts. No other single group of fecal microflora differed in the scouring and non-scouring animals. The composition of the early-weaning ration offered to the animals did not, in itself, influence the fecal microflora to any appreciable extent. In another series of experiments, enumeration of coliforms and lactobacilli was conducted on samples obtained from different segments of the intestinal tracts of scouring and non-scouring pigs. Increased numbers of coliforms and decreased numbers of lactobacilli were observed at all levels of the intestinal tracts of the scouring animals. However, these changes were more marked in the duodenal samples than in those obtained from other parts of the intestine.     

低出生体重儿肠道菌群及肠道屏障功能的研究

目的研究低出生体重儿的肠道菌群分布情况和肠道屏障功能的变化。方法以低出生体重儿(1 500g≤体重2 500g)为研究对象,采用16SrRNA荧光定量PCR技术和JY-DLT肠道屏障功能分析系统检测低出生体重儿出生后第7天粪便中双歧杆菌、乳杆菌、大肠埃希菌、肠球菌4种细菌的含量以及血清中的二胺氧化酶、D-乳酸和细菌内毒素的浓度,比较正常新生儿与低出生体重儿肠道菌群和肠道屏障功能的差异,分析不同喂养方式、并发症对低出生体重儿肠道菌群及肠道屏障功能的影响。结果 (1)低出生体重儿组粪便中大肠埃希菌、肠球菌、乳杆菌、双歧杆菌含量均明显低于健康新生儿组(P0.05),血清中二胺氧化酶、D-乳酸高于健康新生儿组(P0.05),细菌内毒素水平差异无统计学意义(P0.05)。(2)母乳喂养组低出生体重儿粪便中双歧杆菌和乳杆菌含量明显高于乳制品喂养组(P0.05),且血清中二胺氧化酶和和D-乳酸含量低于乳制品喂养组(P0.05),细菌内毒素水平差异无统计学意义(P0.05)。(3)无并发症组低出生体重儿粪便中乳杆菌和双歧杆菌含量明显高于有并发症组(P0.05),其血清中二胺氧化酶、D-乳酸和细菌内毒素水平均低于有并发症的低出生体重儿(P0.05)。结论低出生体重儿的肠道菌群和肠道屏障功能都与正常新生儿存在差异,母乳喂养有助于肠道有益菌的定植和肠道屏障功能的恢复。     

Fate of endotoxin in the intestine of newborn bacteria-free piglets monocontaminated withEscherichia coli

The question of the mechanism by which the Gram-negative intestinal microflora acts as an antigenic stimulus for the development of immune reactions in the newborn was studied. Using newborn bacteria-free piglets artificially monocontaminated withE coli P-1 as the model, it was determined whether somatic antigens are released and absorbed in the intestine and infiltrate into the organism. The presence of specific antigen was determined by haemagglutination inhibition, 2–13 days after contamination, in organ homogenates (liver, spleen, mesenteric nodes, kidney) extracted with water and phenol. The results show that specific antigen was not demonstrated by haemagglutination inhibition in any of the test organs of newborn piglets contaminated per os with a living strain ofE. coli. In the control group, to which a large dose of purified endotoxin (1–10 mg./kg.) was administered into the lumen of the intestine or into the blood stream, the findings in the organs were positive. This shows that if a sufficient amount of endotoxin is present in the intestine, it is resorbed and can be demonstrated by the given technique, particularly in the liver and mesenteric nodes.     

Parameters and techniques to determine intestinal health of poultry as constituted by immunity,integrity, and functionality

The intestinal microflora lives in intimate contact with its surrounding intestinal wall and the bacteria can exert beneficial or deleterious effects on the host, depending on whether they are classified as probiotics or as pathogens. The interaction is determined on one hand by characteristics of the microorganisms, and on the other hand by characteristics of the intestinal wall. Together they determine the health status of the intestine. This review describes parameters and techniques (with advantages and disadvantages) available for poultry to identify the characteristics of the intestinal health, as constituted by three components: immunity, integrity, and functionality. To investigate intestinal immunity, in situ detection of various cell populations of the immune system with specific monoclonal antibodies using immunocytochemical staining is a reliable, semi-quantitative method. In vitro assays to measure functional aspects of T lymphocytes, B lymphocytes, plasma cells, natural killer cells, macrophages, and phagocytes are applicable to intestinal wall tissue. For investigation of intestinal integrity, in situ detection of villous height and crypt depth and their ratio, and villus arrangement is still an easy, routine histological method. In addition, expression levels of specific molecules, such as E-cadherin, different growth factors, and trefoil factor, seem promising parameters. To investigate functionality of the intestine, the permeability can be measured as the rate of transport of tracer molecules across the epithelial surface. Furthermore, determination of the level of mucus secretion and its composition are a valuable tool. These parameters for immunity, integrity and functionality, or a combination thereof, are indispensable to investigate the influence of intestinal microorganisms on intestinal health.     

Clinical and diagnostic importance of the evaluation of the Ig proteases activity in children with intestinal dysbacteriosis

The specific activity of serine, metal dependent and thiolic Ig proteases in the coprofiltrates of children with manifestations of intestinal dysbacteriosis was determined by the enzyme immunoassay. 56 children with pronounced symptoms of intestinal disorders (37 children aged up to 1 year and 19 children over 1 year) were examined. A group of 25 clinically healthy children was used as control. Simultaneously with protease activity of coprofiltrates, there was detected the level of Ig-degrading activity of the opportunistic bacteria islolates of different taxonomic groups from feces of children with dysbacteriosis of different severity (as determined by the classical bacteriological method). The evaluation of the Ig-proteolytic activity of fecal supernatants, associated with the presence of serine, metal-dependent and thiolic proteases in the intestine, as well as detection of such proteases in microbial isolates, seems to be highly important for the diagnosis of intestinal disorders in children and is recommended for screening of intestinal dysbacteriosis.     

Intestinal microbial patterns of the common marmoset and rhesus macaque

The intestinal microflora of common marmosets and rhesus monkeys were compared by enumerating bacteria from the small and large intestines. Rhesus monkeys had a consistent microflora pattern manifest by higher concentrations of total and Gram-negative aerobic and facultatively anaerobic bacteria, as well as aerobic and anaerobic Lactobacilli, in the large intestine as compared to the small intestine. In contrast, the marmoset microflora were considerably more variable. Approximately two-thirds of the marmosets (designated group A) had an overall profile that resembled the rhesus monkeys, but they had significantly higher concentrations of Gram-negative microflora in their large intestines than the rhesus monkeys. The remaining marmosets (group B) had higher concentrations of bacteria in the small intestine as compared to the large intestine, with the large intestinal concentrations being significantly lower than in the rhesus monkeys and group A marmosets. Moreover, the marmosets did not have detectable levels of aerobic Lactobacilli, and anaerobic Lactobacilli concentrations were significantly lower than in the rhesus macaques. Although it is unknown why microflora differ across species, it is likely that evolutionary adaptations in anatomy and functioning of the gastrointestinal tract influence the concentration and types of bacteria residing as the normal intestinal microflora.     

Structural Shifts of Fecal Microbial Communities in Rats with Acute Rejection after Liver Transplantation

Bacterial translocation and the development of sepsis after orthotopic liver transplantation (OLT) may be promoted by immunological damage to the intestinal mucosa or by quantitative and qualitative changes in intestinal microbiota. This study monitored structural shifts of gut microbiota in rats with OLT using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR (RT-qPCR). RT-qPCR targets six major microorganisms (Domain Bacteria, Bacteroides, Bifidobacteria, Enterobacteriaceae, Lactobacillus and Clostridium leptum subgroup). Isograft, Allograft and Sham model were studied. Bacterial translocation to host organs and plasma endotoxin were determined. Alteration in gut microbiota was associated with the elevation of plasma endotoxin and a higher rate of bacterial translocation (BT) to liver in rats with acute rejection. Dynamic analysis of DGGE fingerprints showed that the gut microbiota structure of animals in the three groups was similar before the operation. But significant alterations in the composition of fecal microbiota in Allograft group were observed at 1 and 2 weeks after the OLT. The acute rejection was accompanied by the shifts of gut microbiota towards members of Bacteroides and Ruminococcus. Results from RT-qPCR indicated that Bacteroides significantly increased at 2 weeks after the OLT, whereas numbers of Bifidobacterium spp. decreased at 1 week and recovered at 2 weeks after the OLT. In summary, our data showed that rats with acute rejection after OLT exhibited significant structure shifts in the gut microbiota which dominant by overgrowth of Bacteroides and Ruminococcus, and these were associated with elevation of plasma endotoxin and higher rate of BT.     

虎斑颈槽蛇肠道微生物组成和分布特征

蛇类是一种研究能量平衡的模型动物,而肠道微生物则帮助宿主获取能量和营养。大多数肠道微生物的研究集中在圈养动物上,而对于野生蛇类的研究很少。本研究从3只野生虎斑颈槽蛇（Rhabdophis tigrinus）的大肠、小肠和泄殖腔内容物提取微生物总DNA,进行16S rRNA基因V4区域扩增子测序,分析虎斑颈槽蛇肠道核心微生物组成和分布特征。结果表明,阿尔法指数在不同肠道区域的多样性不存在显著性差异。大肠、小肠和泄殖腔3个肠道部位共享534个操作分类单元（OTUs）,大肠包含最多特有OTUs（388个）。门水平,梭杆菌门（Fusobacteria）在小肠（52.87% ± 14.49%）、大肠（41.12% ± 22.60%）和泄殖腔（65.70% ± 10.44%）均为优势菌。属水平,鲸杆菌属（Cetobacterium）在小肠（46.36% ± 13.86%）、大肠（21.95% ± 9.82%）和泄殖腔（58.18% ± 14.29%）为优势菌,柠檬酸杆菌属（Citrobacter）在小肠和泄殖腔中有显著性差异。此外,在其肠道检测到很多潜在的致病菌,例如,柠檬酸杆菌属、明串珠菌属（Trichococcus）和丹毒丝菌属（Erysipelothrix）,了解了潜在致病菌在虎斑颈槽蛇肠道中的分布情况。在聚类分析中发现泄殖腔的数据重复性较好,将本研究泄殖腔数据与前人研究过的食鱼蝮（Agkistrodon piscivorus）泄殖腔高通量测序数据进行联合分析,为更广泛开展不同蛇类肠道微生物组成的差异性分析提供理论借鉴依据。     

Rapid diagnosis of dysbacteriosis by detecting beta-aspartylglycine in the feces in an experiment

In experiments on Fischer rats (F-344), both with common microflora and germ-free, the influence of the systemic destination of different antibiotics (tetracycline + ampicillin, gentamicin + kefzol, gentamicin, fradizine) on the intestinal microflora, the content of beta-ospartylglycine in feces and the colonization resistance of the intestinal tract to Staphylococcus aureus B-243 and Pseudomonas aeruginosa No. 93 has been studied. The early appearance of beta-aspartyl-glycine in the supernatant of fecal samples has been shown to be the first sign of dysbacteriosis and to indicate the decrease of the colonization resistance of the intestine to opportunistic bacteria.