作者想感谢所有欧洲投资银行的成员，为开发这一技术。这项研究得到了部分科研优先领域"交通膜"（议员）和感染现象"黑客帝国"（KH），青年科学家（SF和KH），科研（何的格兰特在援助）和"细胞内物流"（何）从教育部，文化，体育，科学和技术日本，创新领域的科学研究。

1。细菌细胞的制备<ol><li>摆放着数甘油荧光的细菌（如绿色荧光蛋白表达的鼠伤寒沙门氏菌）的LB琼脂平板，含100μg / ml的氨苄青霉素。 </li><li>文化从单菌落LB琼脂2毫升的新的LB培养基中过夜。 </li><li>添加0.5毫升细菌培养，直到在600 nm的光密度达到1.0至4.5毫升的新的LB培养基孵育。 </li><li>收获的细菌细胞，离心（3000 XG，5分钟，4℃）。 </li><li>弃上清，5.0毫升的无菌磷酸盐缓冲液（PBS）洗两次。 </li><li> 5毫升的PBS重悬菌体，并用50μL暂停接种含有约10 7菌落形成单位（CFU） 。 </li><li>在使用荧光标记的细菌的情况下，细菌细胞标记的荧光标记试剂，根据标准协议。 </li></ol> 2。一nesthesia <ol><li> 5％（V / V）填充一个小塑料盒（10 × 10 × 5厘米），蒸发异氟醚（流量:200毫升/分钟）与空气混合。 </li><li>麻醉八个弱十六岁的男性或女性在框中小鼠。 </li><li>麻醉后，将小鼠进行尸检表。 </li><li>不断麻醉2％（V / V）的小鼠异氟醚蒸发与空气混合（流量:200毫升/分钟）（ 图1）。这是一个终端程序。 </li></ol> 3。结扎Peyer氏补丁循环检测<ol><li>腹部皮肤，然后切开1厘米削减麻醉小鼠的腹腔腹膜，取出小肠Peyer氏补丁。 </li><li>结扎肠道缝纫纱，照顾，以避免血管。注:只能绑定一个侧面的肠道，并留下对方松动。 </li><li>宽松的小号50μL菌悬液或PBS（对照组）用注射器注入结扎Peyer氏补丁循环肠道IDE（ 图2）。 </li><li>绑定宽松的一面，并关闭鼠标的腹部剪辑。 </li><li> 1小时后，取出结扎Peyer氏补丁循环，安乐死颈椎脱位鼠标。 </li><li>海关Peyer氏补丁结扎Peyer氏补丁循环。 </li><li>闪烁的PBS1毫升Peyer氏补丁根尖使用注射器针，去除多余的粘膜液体和细菌。一个额外的两次闪烁Peyer氏补丁。 </li></ol> 4。 Peyer氏补丁整装染色和激光共聚焦显微镜分析<ol><li> Peyer氏补丁修复BD Cytofix / Cytoperm冰1小时解决方案。 </li><li>屋宇署权限/洗5分钟缓冲1毫升Peyer氏补丁洗净三次和然后块1毫升阻止包含0.1％（W / V）皂甙，0.2％（W ​​/ V）BSA的PBS 30分钟就缓冲冰。 </li><li>加入200倍稀释的抗鼠GP2的单克隆抗体（5微克/毫升）标本检测M细胞。 </li><li>在室温下2小时或过夜孵育4 ° C。 </li><li> 1毫升冷PBS洗三次，再加入200倍稀释的抗鼠IgG抗体DyLight549（20微克/毫升）的标本共轭。 </li><li>孵育2小时的冰样本。 </li><li> 1毫升冷PBS洗三次，然后加入50倍稀释Alexa的633标记的鬼笔环肽标本检测F -肌动蛋白。 </li><li>在冰上孵育2小时。 </li><li> 1毫升冷PBS轻轻冲洗。然后放置在三到四个圆形玻璃盖片，幻灯片和嵌入的标本中30％的甘油PBS溶液幻灯片上（ 图3）。 </li><li>一个DM - IRE2共聚焦激光扫描显微镜或DeltaVision恢复反卷积显微镜观察标本。 </li></ol> 5。代表性的成果: 鼠标的标本例如结扎Peyer氏修补与绿色荧光蛋白伤寒的循环实验murium观察与DeltaVision恢复反卷积显微镜（ 图4）。 GFP的鼠伤寒沙门氏菌是transcytosed由GP2 + M细胞。可以由M细胞transcytosed的小鼠Peyer氏修补肠袢检测可以识别的一种共生或致病菌。 <img alt="图1" src="/files/ftp_upload/3225/3225fig1.jpg" /> 图1。蒸发异氟醚条件下小鼠的麻醉。汽化异氟醚麻醉专用器具（左侧）提供。 <img alt="图2" src="/files/ftp_upload/3225/3225fig2.jpg" /> 图2。结扎Peyer氏修补肠袢实验总结。荧光菌悬液通过注射器注入肠道松边结扎Peyer氏补丁循环。 <img alt="图3" src="/files/ftp_upload/3225/3225fig3.jpg" /> <s聪&gt;图3。安装的标本。嵌入式标本中30％的甘油在PBS溶液中，在一个特殊的幻灯片，在其中一个穿孔的塑料板（1mm厚）粘合剂载玻片上的约束。 <img alt="图4" src="/files/ftp_upload/3225/3225fig4.jpg" /> 图4。结扎Peyer氏修补与GFP的鼠伤寒沙门氏菌循环实验鼠标的例子。 GP2的，这是M细胞特异性标志物5报道，染色试样抗鼠GP2的抗体（红色）。与DeltaVision恢复反卷积显微镜观察。 GFP的表达鼠伤寒沙门氏菌是共同本地化与GP2的M细胞的顶质膜和subapical细胞质囊泡（箭头） 。顶，根尖的看法。底部和侧面，外侧意见。比例尺:10微米。

<ol>
	<li>Fagarasan, S., Honjo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+IgA+synthesis:+regulation+of+front-line+body+defences.">Intestinal IgA synthesis: regulation of front-line body defences.</a> Nat. Rev. Immunol. 3, 63-72 (2003).</li><li>Hapfelmeier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+microbial+colonization+of+germ-free+mice+reveals+the+dynamics+of+IgA+immune+responses.">Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses.</a> Science. 328, 1705-1709 (2010).</li><li>Owen, R. L., Jones, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+specialization+within+human+Peyer's+patches:+an+ultrastructural+study+of+intestinal+lymphoid+follicles.">Epithelial cell specialization within human Peyer's patches: an ultrastructural study of intestinal lymphoid follicles.</a> Gastroenterology. 66, 189-203 (1974).</li><li>Neutra, M. R., Mantis, N. J., Kraehenbuhl, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collaboration+of+epithelial+cells+with+organized+mucosal+lymphoid+tissues.">Collaboration of epithelial cells with organized mucosal lymphoid tissues.</a> Nat. Immunol. 2, 1004-1009 (2001).</li><li>Hase, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+through+glycoprotein+2+of+FimH(+)+bacteria+by+M+cells+initiates+mucosal+immune+response.">Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response.</a> Nature. 462, 226-230 (2009).</li><li>Punyashthiti, K., Finkelstein, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteropathogenicity+of+Escherichia+coli.+I.+Evaluation+of+mouse+intestinal+loops.">Enteropathogenicity of Escherichia coli. I. Evaluation of mouse intestinal loops.</a> Infect. Immun. 4, 473-478 (1971).</li><li>Owen, R. L., Pierce, N. F., Apple, R. T., Cray, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M+cell+transport+of+Vibrio+cholerae+from+the+intestinal+lumen+into+Peyer's+patches:+a+mechanism+for+antigen+sampling+and+for+microbial+transepithelial+migration.">M cell transport of Vibrio cholerae from the intestinal lumen into Peyer's patches: a mechanism for antigen sampling and for microbial transepithelial migration.</a> J. Infect. Dis. 153, 1108-1118 (1986).</li><li>Owen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M+cells--entryways+of+opportunity+for+enteropathogens.">M cells--entryways of opportunity for enteropathogens.</a> J. Exp. Med. 180, 7-9 (1994).</li><li>Clark, M. A., Hirst, B. H., Jepson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M-cell+surface+beta1+integrin+expression+and+invasin-mediated+targeting+of+Yersinia+pseudotuberculosis+to+mouse+Peyer's+patch+M+cells.">M-cell surface beta1 integrin expression and invasin-mediated targeting of Yersinia pseudotuberculosis to mouse Peyer's patch M cells.</a> Infect. Immun. 66, 1237-1243 (1998).</li><li>Jensen, V. B., Harty, J. T., Jones, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+the+invasive+pathogens+Salmonella+typhimurium,+Listeria+monocytogenes,+and+Shigella+flexneri+with+M+cells+and+murine+Peyer's+patches.">Interactions of the invasive pathogens Salmonella typhimurium, Listeria monocytogenes, and Shigella flexneri with M cells and murine Peyer's patches.</a> Infect. Immun. 66, 3758-3766 (1998).</li><li>Nagai, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Peyer's+patches+in+the+induction+of+Helicobacter+pylori-induced+gastritis.">Role of Peyer's patches in the induction of Helicobacter pylori-induced gastritis.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 8971-8976 (2007).</li></ol>

没有利益冲突的声明。

<html>结扎Peyer氏菌悬液的补丁的孵化时间通常为1小时观察到M细胞的细菌掺入。在4小时孵化的情况下，细菌往往检测Peyer氏斑的T细胞区。由于蒸发异氟醚吸入麻醉小鼠保持稳定，结扎Peyer氏菌悬液的补丁的孵化时间可延长至观察T细胞Peyer氏补丁区移动的细菌。这个实验的重要的一点是要小心，不要划伤血管结扎肠道时。在不使用GFP表达的细菌的情况下，我们也可以选择使用商业荧光标记试剂荧光标记的细菌。如果试剂可能抑制细菌，你要使用的，附件M细胞上表达的分子，你应该使用特定的抗体，如果有的话，检测纳入细菌。 ontent"的结扎循环检测方法以前描述6，7我们改善如下一些这种方法:。。。1异氟醚是一个保持小鼠稳定2的麻醉剂使用约1厘米结扎肠袢包括Peyer氏。补丁是成立3。M细胞在细菌是荧光由荧光标签的试剂或过度表达如GFP荧光蛋白标记。4。的卵泡，相关的覆盖Peyer氏补丁上皮M细胞是检测整体的贴装immunostainig与反GP2抗体5。M细胞的荧光细菌transcytosis共聚焦显微镜分析观察。 我们和其他人表现出不同的异种抗原，积极争取进入M细胞。相当数量的病原体， 包括沙门氏菌，志贺氏菌，耶尔森菌属。利用主机入侵8-10 M细胞。此外，球形体的Helicobacter幽门螺杆菌可能转运到Peyer氏通过M细胞的补丁，这种易位是在胃的慢性炎症11感应。另一方面，这些病原体侵入M细胞的机制，仍有待完全阐明。我们建议结扎Peyer氏补丁循环检测解剖M细胞在体内的病原体之间的相互作用是一个合适的的实验方法。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> LB琼脂氨苄青霉素 100 </td><td>西格玛</td><td> L5667 </td><td></td></tr><tr><td> Cy3标记单反应 染料包</td><td> GE医疗集团</td><td> PA23001 </td><td></td></tr><tr><td>的Alexa Fluor 350 羧酸， 琥珀酰亚胺酯</td><td> Invitrogen公司</td><td> A10168 </td><td></td></tr><tr><td>吸入麻醉 仪器</td><td>信浓制作所</td><td> SN - 487 </td><td></td></tr><tr><td>固定和 通透 解决方案</td><td>屋宇署</td><td> 554722 </td><td></td></tr><tr><td>抗鼠 糖蛋白2 抗体</td><td> MBL </td><td>D278 - 3 </td><td> 200倍稀释液（5μg/ml） </td></tr><tr><td>羊抗鼠IgG， F（AB&#39;）2片段 具体</td><td>杰克逊 ImmunoResearch </td><td> 112-505-006 </td><td> 200倍稀释液（20μg/ml） </td></tr><tr><td>的Alexa Fluor 633 鬼笔环肽</td><td>分子探针</td><td> A22284 </td><td> 50倍稀释</td></tr><tr><td>激光共聚焦 扫描显微镜</td><td>徕卡显微系统</td><td> DMIRE2 </td><td></td></tr><tr><td> DeltaVision 恢复 反卷积 显微镜</td><td>应用精密</td><td> DeltaVision核心</td><td></td></tr></tbody></table>

application of a mouse ligated peyer&#x2019;s patch intestinal loop assay to evaluate bacterial uptake by m cells

我们的肠道内居住的共生细菌的数量巨大。对胃肠道的黏膜表面不断暴露给他们，并偶尔病原体。肠道相关淋巴组织（GALT）为诱导黏膜免疫反应，这些微生物1，2中发挥了关键作用。要启动黏膜免疫反应，粘膜抗原必须运从肠腔整个组织，如Peyer氏斑淋巴滤泡的上皮屏障。这介导的抗原transcytosis是由专门的上皮M细胞3， 4。 M细胞是非典型的上皮细胞，积极吞噬大分子物质和微生物。 M细胞与树突状细胞（DCs）和巨噬细胞，靶抗原降解的溶酶体，主要transcytose内在抗原。这种积极通过M细胞的大分子transcytosis提供抗原的根本组织淋巴滤泡D是认为启动抗原特异性黏膜免疫反应的关键。然而，促进M细胞抗原摄取的分子机制在很大程度上是未知的。我们曾报道，糖蛋白2（GP2），特别是肠之间的M细胞的根尖质膜上表示，作为一个transcytotic受体的共生和致病肠杆菌的一个子集，包括大肠杆菌和沙门氏菌血清型鼠伤寒沙门氏菌（鼠伤寒沙门氏菌），通过承认FimH，细菌外膜5菌毛类型的组成部分，我。在这里，我们提出了一个鼠标Peyer氏修补肠袢的检测，以评估M细胞的细菌摄取的应用方法。这个方法是鼠标肠袢先前所描述的6，7含量的改进版本。改进的要点如下:1。异氟醚被用作麻醉剂。 2。约1厘米结扎肠循环includING Peyer氏补丁成立。 3。 M细胞的细菌荧光标记的荧光标记试剂或过度表达的荧光蛋白，如绿色荧光蛋白（GFP）。 4。 M细胞相关的卵泡上皮覆盖Peyer氏补丁与反GP2抗体检测整个安装immunostainig。 5。荧光共聚焦显微镜分析观察M细胞的细菌transcytosis。小鼠Peyer氏补丁肠袢实验可以提供答案，什么样的共生或transcytosed M细胞的致病细菌，并可能导致我们了解如何通过M细胞刺激粘膜免疫系统的分子机制。

Watch this Scientific Journal Video about Application of a Mouse Ligated Peyerâ€™s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells at JoVE.com

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

在一个专门的卵泡相关Peyer氏斑上皮M细胞在肠道相关淋巴组织的粘膜免疫监视中发挥的重要作用。在这里，我们描述了细菌transcytosis M细胞的评价方法<em&gt;在体内</em&gt;。这种方法提供了一种方法，以了解在免疫系统中的M -细胞功能。

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

application-mouse-ligated-peyers-patch-intestinal-loop-assay-to

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

19.4K 次观看.  RIKEN Research Center for Allergy and Immunology. 在一个专门的卵泡相关Peyer氏斑上皮M细胞在肠道相关淋巴组织的粘膜免疫监视中发挥的重要作用。在这里，我们描述了细菌transcytosis M细胞的评价方法在体内。这种方法提供了一种方法，以了解在免疫系统中的M -细胞功能。

视频: Application of a Mouse Ligated Peyer’s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

隔离和免疫淋巴细胞和树突状细胞的小鼠Peyer氏斑

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

科研

免疫与感染

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

益生菌酵母的转型及其胃肠道粘膜免疫组织恢复继小鼠口服灌胃

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

小鼠小肠免疫系统淋巴细胞的分离

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

人类干细胞衍生的伊拉尔肠细胞单层中M细胞样细胞的诱导分化

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

基于内显微镜的方法，用于评估肠道渗透性和上皮细胞脱落性能

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

使用标准化肠道循环模型对小鼠肠道渗透性和中性粒细胞转导的功能评估

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important phenomenon in the gut immunity. The stroma of secondary lymphoid organs, such as Peyer&#8217;s patches (PPs) or mesenteric lymph nodes, are where na&#239;ve lymphocytes sense antigens. Na&#239;ve lymphocytes circulate through the bloodstream to reach high endothelial venules, the portal of entry into PPs. Some immunomodulators are estimated to influence lymphocyte migration, but the precise evaluation of microcirculation dynamics is very difficult, and establishing a method to observe lymphocyte migration in vivo can contribute to the clarification of the precise mechanisms. We refined the method of collecting lymphocytes from the lymph duct and observing the detailed dynamics of gut-tropic lymphocytes in rat PPs. We chose confocal laser scanning microscopy to observe rat PPs in vivo and recorded it using time-lapse photography. We can now obtain clear images that can contribute to the analysis of lymphocyte dynamics.

Here, we describe a precise method for collecting thoracic duct lymphocytes and observing the migration of gut-tropic lymphocytes in rat Peyer&#8217;s patches for 3 hours using time-lapse photography. This technique can clarify how the dynamics of lymphocytes are affected under inflammatory conditions.

鼠佩耶斑块中淋巴细胞动力学的微观观察

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important ...

医学

<meta charset="utf8"></head><body>通过肠道作研究术后肠梗阻的小鼠模型

Postoperative Ileus Murine Model

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI is a consequence of mechanical damage during surgery, resulting in disruption of motility in the gastrointestinal tract. The mechanisms of POI are related to aberrant neuronal sensitivity, impaired epithelial barrier function, and increased local inflammation. However, the details remain enigmatic. Therefore, experimental murine models are crucial for elucidating the pathophysiology and mechanism of POI injury and for the development of novel therapies.
Here, we introduce a murine model of POI generated via intestinal manipulation (IM) that is similar to clinical surgery; this is achieved by mechanical damage to the small intestine by massaging the abdomen 1-3 times with a cotton swab. IM delayed gastrointestinal transit 24 h after surgery, as assessed by FITC-dextran gavage and fluorescence detection of the segmental digestive tract. Moreover, tissue swelling of the submucosa and immune cell infiltration were investigated by hematoxylin and eosin staining and flow cytometry. Proper pressure of the IM and a hyperemic effect on the intestine are critical for the procedure. This murine model of POI can be utilized to study the mechanisms of intestinal damage and recovery after abdominal surgery.

<meta charset="utf8"></head><body>术后肠梗阻鼠模型

We describe a murine model of postoperative ileus generated via intestinal manipulation. Gastrointestinal transit function, pathologic changes, and immune cell activation were assessed 24 h after surgery.

术后肠梗阻鼠模型

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI ...

鼠标应用结扎Peyer氏修补肠袢含量评价M细胞的细菌吸收

摘要

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此视频中的章节

隔离和免疫淋巴细胞和树突状细胞的小鼠Peyer氏斑

益生菌酵母的转型及其胃肠道粘膜免疫组织恢复继小鼠口服灌胃

小鼠小肠免疫系统淋巴细胞的分离

人类干细胞衍生的伊拉尔肠细胞单层中M细胞样细胞的诱导分化

基于内显微镜的方法，用于评估肠道渗透性和上皮细胞脱落性能

使用标准化肠道循环模型对小鼠肠道渗透性和中性粒细胞转导的功能评估

鼠佩耶斑块中淋巴细胞动力学的微观观察

术后肠梗阻鼠模型

术后肠梗阻鼠模型

术后肠梗阻鼠模型