एक माउस के आवेदन Peyer पैच आंतों लूप परख ligated एम कोशिकाओं द्वारा बैक्टीरियल तेज मूल्यांकन
Summary
एम में एक विशेष कूप जुड़े Peyer पैच कवर उपकला कोशिकाओं पेट जुड़े lymphoid ऊतक में mucosal immunosurveillance के लिए एक महत्वपूर्ण भूमिका निभाते हैं. यहाँ हम बैक्टीरियल transcytosis के लिए एम कोशिकाओं द्वारा मूल्यांकन पद्धति का वर्णन<em> Vivo में</em>. इस पद्धति का एक प्रतिरक्षा प्रणाली में एम सेल समारोह को समझने के लिए एक तरीका प्रदान करता है.
Abstract
The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer’s patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer’s patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer’s patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer’s patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer’s patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.
Protocol
Discussion
जीवाणु निलंबन के साथ ligated है Peyer पैच के ऊष्मायन समय 1 घंटे के लिए आम तौर पर एम कोशिकाओं में बैक्टीरिया निगमन का पालन करने के लिए. 4 बजे ऊष्मायन के मामले में, जीवाणु अक्सर Peyer पैच की टी सेल के क्षेत्र में पाया ज…
Declarações
The authors have nothing to disclose.
Acknowledgements
लेखकों के लिए इस तकनीक के विकास के लिए सभी EIB सदस्यों को धन्यवाद देना चाहूंगा. इस शोध में भाग प्राथमिकता क्षेत्रों "झिल्ली आवागमन (हो), और संक्रामक घटना की मैट्रिक्स '(के.एच.), युवा वैज्ञानिकों (एस एफ और के.एच.), और वैज्ञानिक अनुसंधान (हो पर वैज्ञानिक अनुसंधान के लिए अनुदान सहायता के द्वारा समर्थित किया गया था ), और अभिनव (हो) से शिक्षा मंत्रालय, संस्कृति, खेल, विज्ञान और प्रौद्योगिकी जापान के "Intracellular रसद" क्षेत्रों पर वैज्ञानिक अनुसंधान.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| LB Agar Ampicillin- 100 |
SIGMA | L5667 | |
| Cy3 mono-Reactive Dye Pack |
GE Healthcare | PA23001 | |
| Alexa Fluor 350 carboxylic acid, succinimidyl ester |
Invitrogen | A10168 | |
| Inhalation anesthesia apparatus |
Shinano Seisakusho | SN-487 | |
| Fixation and Permeabilization Solution |
BD | 554722 | |
| Anti mouse Glycoprotein 2 antibody |
MBL | D278-3 | 200-fold dilution (5μg/ml) |
| Goat Anti-Rat IgG, F(ab’)2 fragment specific |
Jackson ImmunoResearch |
112-505-006 | 200-fold dilution (20μg/ml) |
| Alexa Fluor 633 Phalloidin |
Molecular Probes | A22284 | 50-fold dilution |
| Confocal laser scanning microscope |
Leica Microsystems | DMIRE2 | |
| DeltaVision Restoration deconvolution microscope |
Applied Precision | DeltaVision Core |
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