利用生物发光活体成像技术观察肠黏膜损伤状态下细菌易位的初步研究

[目的]临床上各种疾病会导致肠黏膜屏障损伤并伴随大量的细菌易位发生。本实验拟使用发光细菌的小鼠活体成像技术,观察肠黏膜损伤状态下易位细菌的动态移位过程。[方法]构建重组荧光脂酶基因(Lux)的质粒pXen-1、pXen-18,转化大肠杆菌DH5a,筛选阳性细菌克隆,扩增培养后,定量(107)注射到肠黏膜屏障损伤模型小鼠肠腔不同部位内,利用生物发光活体成像技术进行细菌易位的病理生理观察研究。实验分为四组:小肠注射菌实验组和对照组、盲肠注射菌实验组和对照组。其中实验组为肠道缺血再灌注损伤,对照组无任何干预措施。[结果]发光细菌小鼠活体成像观察到,存在肠黏膜损伤的小鼠肠腔内的细菌数量及分布发生明显的改变,提示细菌易位在肠黏膜损伤时起着重要的病理生理作用。注入小肠内的发光细菌数量在试验过程中明显下降,提示小肠肠腔环境可以杀灭一定的细菌。[结论]生物发光活体成像技术,可以直观、可靠、敏感地观察肠道内细菌的存活状态,并可观察肠黏膜损伤状态下细菌易位的动态过程。该技术为肠黏膜屏障损伤细菌易位机制的研究提供了一个有力的工具。     

肠屏障功能障碍

1肠屏障相关概念 1.1 肠屏障是指肠道能防止肠腔内的有害物质如细菌和毒素穿过肠黏膜进入体内其他组织器官和血液循环的结构和功能的总和.它包括:肠黏膜上皮、肠黏液、肠道菌群、分泌性免疫球蛋白、肠道相关淋巴组织(GALT)、胆盐、激素和胃酸等[1～3].一般将其归纳为三部分:①生态屏障即肠道内的常居菌群;②机械屏障即黏膜上皮;③免疫屏障即黏膜细胞的分泌性IgA、黏膜内及黏膜下各种免疫细胞(包括巨噬细胞、中性粒细胞、NK细胞、淋巴细胞-即IELs)、派尔集合淋巴结和肠系膜淋巴结(MLN).其中小肠黏膜上皮细胞非常重要,它在充满细菌和毒素的肠腔和其他组织及血循环之间构建了一个物理性屏障,一般意义上的肠屏障即指这层结构[3].     

全肠外营养对肠道免疫功能影响的研究

本研究采用ＳＰＦ大鼠ＴＰＮ支持模型，观察标准ＴＰＮ和改良ＴＰＮ不同营养模式对肠免疫屏障功能的影响，探讨ＴＰＮ导致肠粘膜免疫抑制的机理，进一步阐述ＴＰＮ导致肠湖泊性感染发生的综合机理。结果显示，标准ＴＰＮ由于缺乏肠粘膜必需氨基酸Ｇｌｎ和肠道刺激，引起肠粘膜免疫系统改变，肠腔细菌Ｓ－ｌｇＡ包被率下降，导致肠粘膜免疫抑制和肠道微生态紊乱、机械屏障损伤协同，至细菌粘附率和易位率上升。各改良组因添加肠道必需     

肠粘膜屏障与胃肠疾病

肠粘膜屏障主要包括机械屏障、免疫屏障、生物屏障,三者之间相辅相成,其功能主要在于防止肠道内细菌及内毒素移位。胃肠道功能的正常发挥有赖于肠粘膜屏障稳定,腹泻、肠易激综合征、炎症性肠病以及全身炎症综合征等诸多临床疾病均存在不同程度的肠粘膜屏障损伤。本文就肠粘膜屏障构成体系,肠屏障损伤与胃肠道疾病的关系的研究情况作综述。     

Ala—GIn增强机体在全胃肠外营养状态下对失血性休克的耐受性

采用ＳＰＦ大鼠全胃肠外营养（ＴＰＮ）支持和失血性休克创伤模型，以ＴＰＮ，ＴＰＮ液中添加丙酰胺谷氨酰胺（Ａｌａ－ＧＩｎ）和经肠饮食（ＥＮ）三种营养方式支持一周后，造成失血性休克，观察机体因营养方式差异而入于不同肠异屏障功能状态下对再次打击的耐受性，结果显示，标准ＴＰＮ组与ＥＮ相比，血浆二胺氧化酶（ＤＡＯ）水平明显低下，肠固有层（ＬＰ）淋巴细胞和浆细胞，肠上皮内淋巴细胞（ＩＥＬ）及肠腔细菌分泌型ＩｇＡ     

Ala-Gln增强机体在全胃肠外营养状态下对失血性休克的耐受性

采用ＳＰＦ大鼠全胃肠外营养（ＴＰＮ）支持和失血性休克创伤模型，以ＴＰＮ、ＴＰＮ液中添加丙氨酰谷氨酰胺（Ａｌａ┐Ｇｌｎ）和经肠饮食（ＥＮ）三种营养方式支持一周后，造成失血性休克，观察机体因营养方式差异而处于不同肠屏障功能状态下对再次打击的耐受性。结果显示，标准ＴＰＮ组与ＥＮ相比，血浆二胺氧化酶（ＤＡＯ）水平明显低下；肠固有层（ＬＰ）淋巴细胞和浆细胞、肠上皮内淋巴细胞（ＩＥＬ）及肠腔细菌分泌型ＩｇＡ（Ｓ┐ＩｇＡ）包被率明显下降；盲肠粘膜菌群Ｅ．ｃｏｌｉ优势增殖，双歧杆菌／大肠杆菌（Ｂ／Ｅ）比值倒置，肠上皮细菌粘附增多；肠道细菌易位率升高；死亡率（４／１２）高。而Ａｌａ┐Ｇｌｎ组因添加肠道必需氨基酸谷氨酰胺（Ｇｌｎ）前体Ａｌａ┐Ｇｌｎ，各参数接近ＥＮ组，肠屏障储备增加，死亡率下降。提示：标准ＴＰＮ由于缺乏肠粘膜必需氨基酸（Ｇｌｎ）和肠道刺激，严重损伤肠屏障功能，失血性休克更加重损害，可能促发脓毒血症和多器官功能不全（ＭＯＤＳ）。对标准ＴＰＮ进行改良，添加肠粘膜保护剂Ａｌａ┐Ｇｌｎ对肠屏障有较好维持作用。这对临床不能经肠道喂养的围手术期病人进行如何更有效的肠外营养支持有一定指导意义     

腹部辐射致肠粘膜屏障损伤模型的建立

目的根据临床上辐射性肠粘膜屏障损伤防治研究的需要，探讨建立稳定及符合辐射性肠粘膜屏障损伤研究动物模型的方法。方法SD大鼠，分别用8．5Gy或9．5Gy^60Co腹部辐射，光镜、电镜、细菌培养及图象分析进行肠粘膜屏障损伤的评估。结果应用9．5Gy所致的辐射性肠粘膜屏障损害大鼠其肠粘膜形态学、病理学改变，死亡率(33％)，及作为肠粘膜屏障损伤的指标之一的肠系膜淋巴结的细菌易位率均稳定，与临床病人的病情接近。结论本模型适合辐射性肠粘膜屏障损伤(如放射性肠炎)治疗(如TPN等)研究的需要。     

肠道屏障功能

0 引言 肠道屏障功能是指肠道上皮具有分隔肠腔内物质,防止致病性抗原侵入的功能.正常情况下,肠道具有屏障作用,可有效地阻挡肠道内500多种、浓度高达1012个/g的肠道内寄生菌及其毒素向肠腔外组织、器官移位,防止机体受内源性微生物及其毒素的侵害.近年来,随着对肠道屏障功能研究的深入,肠道屏障的概念已不再局限于传统的肠粘膜机械屏障.目前认为,正常肠道屏障功能的维持依赖于肠粘膜上皮屏障、肠道免疫系统、肠道内正常菌群、肠道内分泌及蠕动,其中最关键的屏障是肠粘膜上皮屏障和肠道粘膜免疫屏障.本文作者就近年来对肠道屏障功能的新认识和观点作一综述.     

应激对肠屏障功能的影响

肠屏障功能是肠道的一个重要特征，正常肠屏障功能的维护依赖于肠粘膜上皮，肠道内正常菌群，肠道内分泌物，蠕动，肠相关免疫细胞等，其中最关键的屏障是肠粘膜上皮屏障。在创伤，严重感染，缺血，饥饿等应激状态下，肠屏障功能将发生一系列病理生理的变化，导致肠粘膜萎缩及通透性增大。应激下肠屏障功能改变的机制是多因素的，没有哪一种单一机制能解释肠屏障功能不全的所有方面。作者就应激时肠屏障功能变化的机制作一综述。     

谷氨酰胺在预防细菌易位和肠上皮细胞损伤中的作用

小肠粘膜的损伤是发生细菌易位的主要机制，而谷氨酰胺在肠粘膜结构与屏障功能的维持中起重要作用。本文通过细胞株、活体肠袢水平的观察来了解谷氨酰胺在减轻细菌易位及肠上皮细胞损伤中的作用。１材料和方法从坏死性肠炎（ＮＥＣ）的婴儿体内分离出的大肠杆菌菌落用在Ｃ...     

重症急性胰腺炎时细胞因子与肠源性细菌和内毒素移位的实验研究

目的 探讨重症急性胰腺炎(SAP)时血浆细胞因子与肠道屏障损害后肠源性细笛和内毒素移位的关系。方法 将SD大鼠(清洁级)72只随机分为假手术组(n=36)和SAP组(n=36)。采用胰管内逆行注射4％牛磺胆酸钠溶液的方法制作SAP模型。观察胰腺和回肠的病理改变，动态测定血浆TNF-a、IL-6、IL-10和DAO活性、LPS水平以及腹腔脏器细菌移位率。结果 制模后血浆TNF-a、IL-6水平明显升高，48h达到高峰，IL-106h后才明显升高；血浆DAO活性早期升高，24h后明显降低；LPS水平早期即有明显升高，48h达到高峰；SAP24h脏器细笛移位率明显升高，72h达到58．3％。结论 SAP早期即有细胞因子水平的升高和肠道屏障的损害，细胞因子通过损害肠遗屏障，引起肠源性细菌和内毒素移位；同时，肠源性细菌和内毒素移位又促进细胞因子的大量释放。加重肠黏膜屏障本身的损害，遣成恶性循环，引起SIPS和MODS的发生，两者关系密切。     

Effects of protein malnutrition and endotoxin on the intestinal mucosal barrier to the translocation of indigenous flora in mice

Since protein malnourished or endotoxemic patients are at increased risk of developing nosocomial infections with enteric organisms, we investigated the effects of these risk factors alone and in combination on the intestinal mucosal barrier to bacteria. Protein malnutrition resulted in severe ileal atrophy that was directly related to the length of time the mice were protein malnourished. Although protein malnutrition did not promote bacterial translocation from the gut to systemic organs, the protein-malnourished mice were more susceptible to endotoxin-induced bacterial translocation than normally nourished mice (p less than 0.01). Since the gross epithelial damage documented after endotoxin administration in normally nourished mice was diminished after protein malnutrition, there was no correlation between the gross appearance of the epithelial mucosal barrier and the extent of endotoxin-induced bacterial translocation. These results suggest that the synergistic effect of endotoxin plus protein malnutrition on bacterial translocation is not primarily related to failure of the gut mucosal barrier. Nonetheless, it appears that protein-malnourished mice are less able to clear translocating bacteria than normally nourished mice.     

肠黏膜屏障的研究进展

胃肠黏膜固有屏障正常稳态的维持,对于防止胃肠腔内细菌、食物抗原、酶和化学药物等直接与黏膜裸露面接触引起的相关疾病至关重要.本文就胃肠黏膜固有屏障的关键结构、成分和功能、调节因素等方面研究进展予以综述.     

Exercise for the Diabetic Gut—Potential Health Effects and Underlying Mechanisms

It can be assumed that changes in the gut microbiota play a crucial role in the development of type 2 diabetes mellitus (T2DM). It is generally accepted that regular physical activity is beneficial for the prevention and therapy of T2DM. Therefore, this review analyzes the effects of exercise training on the gut microbiota composition and the intestinal barrier function in T2DM. The current literature shows that regular exercise can influence the gut microbiota composition and the intestinal barrier function with ameliorative effects on T2DM. In particular, increases in the number of short-chain fatty acid (SCFA)-producing bacteria and improvements in the gut barrier integrity with reduced endotoxemia seem to be key points for positive interactions between gut health and T2DM, resulting in improvements in low-grade systemic inflammation status and glycemic control. However, not all aspects are known in detail and further studies are needed to further examine the efficacy of different training programs, the role of myokines, SCFA-producing bacteria, and SCFAs in the relevant metabolic pathways. As microbial signatures differ in individuals who respond differently to exercise training programs, one scientific focus could be the development of computer-based methods for the personalized analysis of the gut microbiota in the context of a microbiota/microbiome-based training program.     

Bile Salt Hydrolase-Competent Probiotics in the Management of IBD: Unlocking the “Bile Acid Code”

Bile acid (BA) species and the gut microbiota (GM) contribute to intestinal mucosa homeostasis. BAs shape the GM and, conversely, intestinal bacteria with bile salt hydrolase (BSH) activity modulate the BA pool composition. The mutual interaction between BAs and intestinal microorganisms also influences mucosal barrier integrity, which is important for inflammatory bowel disease (IBD) pathogenesis, prevention and therapy. High levels of secondary BAs are detrimental for the intestinal barrier and increase the intestinal inflammatory response and dysbiosis. Additionally, a lack of BSH-active bacteria plays a role in intestinal inflammation and BA dysmetabolism. Thus, BSH-competent bacteria in probiotic formulations are being actively studied in IBD. At the same time, studies exploring the modulation of the master regulator of BA homeostasis, the Farnesoid X Receptor (FXR), in intestinal inflammation and how this impacts the GM are gaining significant momentum. Overall, the choice of probiotic supplementation should be a peculiar issue of personalized medicine, considering not only the disease but also the specific BA and metabolic signatures of a given patient.     

一氧化氮与肠屏障功能

肠屏障功能是肠道的一个重要特征,可以保护宿主不受外来抗原的干扰和入侵,它由一系列特异性和非特异性防御机制组成,包括肠管的蠕动、消化液的分泌、肠上皮细胞间的紧密连接以及肠道免疫系统.在烧伤、创伤、失血性休克、放疗、化疗、严重感染等情况下,会相继出现肠屏障破坏、细菌移位,严重者会发生多器官功能障碍.一氧化氮作为一种细胞的小分子物质,在各种生理病理中具有重要作用.近来研究发现,其毒性代谢产物过氧亚硝基阴离子(ONOO-)参与肠黏膜损伤.作者就一氧化氮与ONOO-在肠屏障功能的保护和损伤方面作一综述.     

Use of probiotics in pediatrics: rationale, mechanisms of action, and practical aspects.

The use of probiotics (ingested microbes that can modify intestinal microbial populations in a way that benefit the host) has moved from concept to actual demonstration of specific benefits by specific microorganisms for specific populations. It is increasingly clear that these benefits to the host are mostly mediated by the profound effect that intestinal microflora (microbiota) have on gut barrier function and host immune response. Intestinal bacteria are more numerous than the human cells of the host that harbors them. Despite having many potential pathogens in this microflora, humans do not routinely get infected. It is no coincidence that gut-associated immune tissue constitutes approximately 80% of all immunologically active cells in the human host. The gut interacts with intestinal bacteria, both resident and ingested, to develop protective mechanisms (via improving gut barrier function and immune stimulation for defense) and appropriate, nonexaggerated responses (via immune modulation and immune tolerance) to support host health. The mechanisms of this interaction between host and bacteria are increasingly being unraveled and in great part explain the clinical benefits that have been reported with specific probiotic bacteria by enhancing host defense mechanisms (such as for treatment and prevention of viral diarrhea and reducing risk of necrotizing enterocolitis), mitigating antibiotic-associated diarrhea, and modulating host immune response (such as in allergic disease).     

Gut Microbial Metabolite-Mediated Regulation of the Intestinal Barrier in the Pathogenesis of Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a chronic inflammatory disease. The disease has a multifactorial aetiology, involving genetic, microbial as well as environmental factors. The disease pathogenesis operates at the host–microbe interface in the gut. The intestinal epithelium plays a central role in IBD disease pathogenesis. Apart from being a physical barrier, the epithelium acts as a node that integrates environmental, dietary, and microbial cues to calibrate host immune response and maintain homeostasis in the gut. IBD patients display microbial dysbiosis in the gut, combined with an increased barrier permeability that contributes to disease pathogenesis. Metabolites produced by microbes in the gut are dynamic indicators of diet, host, and microbial interplay in the gut. Microbial metabolites are actively absorbed or diffused across the intestinal lining to affect the host response in the intestine as well as at systemic sites via the engagement of cognate receptors. In this review, we summarize insights from metabolomics studies, uncovering the dynamic changes in gut metabolite profiles in IBD and their importance as potential diagnostic and prognostic biomarkers of disease. We focus on gut microbial metabolites as key regulators of the intestinal barrier and their role in the pathogenesis of IBD.     

创伤性脑损伤后肠黏膜结构和屏障功能的变化

目的:探讨创伤性脑损伤后肠黏膜形态和屏障功能的变化,了解肠黏膜屏障功能障碍的发生时间及其严重程度. 方法:雄性Wistar 大鼠随机分为无脑损伤的对照组和脑损伤后3、12、24、72 h和7天组,每组6只.应用组织病理和电镜观察肠黏膜结构的变化,通过测定血浆内毒素水平和肠黏膜通透性,以评价肠黏膜屏障功能. 结果:创伤性脑损伤后3 h即出现肠黏膜的病理改变,然后逐渐加重.与对照组相比,脑损伤后3、12和24 h血浆内毒素水平明显升高,伤后72 h达到高峰,第7天开始下降.血浆内毒素水平在伤后3 h和72 h出现两个峰值.脑损伤后肠黏膜通透性明显增加. 结论:创伤性脑损伤后3 h即可引起明显的肠黏膜结构和屏障功能损害,至伤后72 h达高峰,此损害可持续7天以上.     

多黏菌素E诱导肠道菌群失调对肠屏障功能和细菌易位的影响

目的:应用多黏菌素E给小鼠灌胃诱导建立肠道菌群失调动物模型,研究肠道菌群失调对肠黏膜屏障和细菌易位的影响。方法:将20只小鼠随机分为两组,每组10只。实验组采用多黏菌素E按0.2 g/kg加入0.2 ml等渗盐水灌胃,1次/d,连续7 d。对照组用等剂量等渗盐水灌胃,1次/d,连续7 d。实验结束次日观察两组小鼠回肠黏膜病理形态、肠道菌群、回肠黏膜组织紧密连接(TJ)蛋白表达和器官(肝、脾、肾、淋巴结)细菌易位率等。结果:实验组小鼠盲肠黏膜和盲肠内容物的肠杆菌数量显著减少。常规病理检查发现,实验组小鼠回肠黏膜充血明显,绒毛稀疏,尖端有少量上皮坏死脱落,与对照组比损伤明显。透射电镜显示,实验组小鼠回肠上皮TJ的电子致密物质明显减少,TJ破坏。回肠黏膜组织中TJ蛋白Claudin-1、Occludin和ZO-1的表达显著下降,器官细菌易位率显著高于对照组。结论:多黏菌素E灌胃能够诱导肠道菌群失调,且导致肠屏障功能损伤和细菌易位。