Generierung von lymphatische Mikropartikel und Nachweis ihrer pro-apoptotische Wirkung auf die Epithelzellen der Atemwege
Summary
Zellmembran-Schuppen-Mikropartikel (MP) aktiv biologischen Vesikel, die isoliert werden können und deren pathophysiologische Effekte in verschiedenen Modellen untersucht. Hier beschreiben wir ein Verfahren zum Erzeugen MPs von T-Lymphozyten (LMPs) abgeleitet und zur Demonstration ihrer proapoptotischen Effekt auf Epithelzellen der Luftwege.
Abstract
Das Interesse an der biologischen Rolle von Zellmembranen abgeleiteten Vesikeln in Zell-Zell-Kommunikation in den letzten Jahren erhöht. Mikroteilchen (MPs) sind eine solche Art der Bläschen, deren Durchmesser von 0,1 um bis 1 um und typischerweise von der Plasmamembran von eukaryotischen Zellen, die Aktivierung oder die Apoptose zu vergießen. Hier beschreiben wir die Erzeugung von T-Lymphozyten abgeleiteten Mikroteilchen (LMPs) von apoptotischen CEM T-Zellen mit Actinomycin D. LMPs werden durch einen mehrstufigen Differenz Zentrifugationsverfahren isoliert und mittels Durchflusszytometrie stimuliert. Dieses Protokoll stellt auch eine in situ Zelltod Nachweismethode für den Nachweis der proapoptotischen Wirkung LMPs auf bronchialen Epithelzellen aus Maus primären Atem bronchiale Gewebeexplantaten abgeleitet. Hierin beschriebene Verfahren eine reproduzierbare Verfahren zur Isolierung reichliche Mengen von LMPs apoptotische Lymphozyten in vitro. LMPs abgeleitetin dieser Weise verwendet werden, um die Eigenschaften der verschiedenen Krankheitsmodellen zu bewerten und für pharmakologische und toxikologische Prüfung. Da das Atemwegsepithel bietet eine Schutz physische und funktionelle Barriere zwischen der äußeren Umgebung und der darunter liegenden Gewebe, stellt die Verwendung von Bronchialgewebe Explantate nicht immortalisierte Epithelzellen Linien ein effektives Modell für Untersuchungen Atemwegstrakt Gewebe erfordern.
Introduction
Microparticles (MPs) are biologically active submicron membrane vesicles released following cell activation or apoptosis. MPs are derived from both healthy and damaged cells and are implicated in many physiological and pathological processes.1 MPs have been detected not only in human plasma, but also in inflammatory and apoptotic tissue. The biological utility of cell membrane–derived MPs has been demonstrated in various settings, including cell signalling models and as pharmacological tools.2,3 We previously demonstrated that LMPs derived from T lymphocytes following actinomycin D stimulation (to induce apoptosis) suppress angiogenesis and inhibit endothelial cell survival and proliferation.4,5 The antiangiogenic effects of LMPs may vary significantly depending on the stimuli used to activate T lymphocytes in vitro.6
The airway epithelium functions as a protective physical and functional barrier. Increased numbers of T lymphocytes in the airway can contribute to cell damage and airway inflammation.7 We have shown that LMPs induce apoptosis of human bronchial epithelial cells,8 which indicated LMPs may change barrier function of bronchial epithelium in vivo. Apoptotic cells can be identified using the TUNEL method, which detects in situ DNA fragmentation.
The overall goal of this protocol is to illustrate the in vitro production of LMPs from a T lymphocyte cell line, and to demonstrate their proapoptotic effect on airway epithelial cells. In situ cell death detection demonstrated that LMPs strongly induce airway bronchial epithelial cell death, suggesting that LMPs-mediated injury to the airway epithelium may impact barrier function of the damaged epithelium.
Protocol
Representative Results
Discussion
Abgeordnete sind aktive Vermittler der interzellulären Übersprechen und deren Studie ist in vielen Bereichen der Wissenschaft verspricht. 11 Diese Studie präsentiert ein detailliertes Protokoll für die in-vitro-Groß Generation LMPs von einem apoptotischen T-Zelllinie abgeleitet. Diese Abgeordneten zum Ausdruck bringen ein großes Repertoire an Lymphozyten-Molekülen und sind biologisch in der Regulation von Zell- und Gewebe Homöostase beteiligt ist. Jedoch kann LMPs aus verschiedenen Quellen ab…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vision-Health Research Network – Diese Arbeit wird durch Zuschüsse aus dem kanadischen Institutes of Health Research (178.918), Fonds de recherche en santé du Québec unterstützt.
Materials
| LMPs production and characterization | |||
| CEM T cells | ATCC | CCL-119 | |
| X-VIVO 15 medium | Cambrex, Walkersville | 04-744Q | |
| Flask T75 | Sarstedt | 83.1813.502 | |
| Flask T175 | Sarstedt | 83.1812.502 | |
| Actinomycin D | Sigma Chemical Co. | A9415-2mg | |
| PBS | Lifetechnologies | 14190-144 | |
| 0.22µm filter | Sarstedt | 83.1826.001 | |
| Annexin-VCy5 | BD Pharmagen | 559933 | |
| FACS flow solution | BD Bio-sciences | 342003 | |
| Fluorescent microbeads (1 um) | Molecular Probes | T8880 | |
| Polysterene counting beads (7 um) | Bangs laboratories | PS06N/6994 | |
| Polypropylene FACS tubes | Falcon | 352058 | |
| 1 ml pipet | Fisher | 13-678-11B | |
| 5 ml pipet | Falcon | 357543 | |
| 25 ml pipet | Ultident | DL-357551 | |
| 1,5 ml conical polypropylene micro tube | Sarstedt | 72.690 | |
| 15 ml conical polypropylene tube | Sarstedt | 62.554.205 | |
| 50 ml conical polypropylene tube | Sarstedt | 62.547.205 | |
| 50 ml high speed polypropylene copolymer tube | Nalgene | 3119-0050 | |
| 250 ml high speed polypropylene bottle | Beckman | 356011 | |
| Protein assay (Bradford assay) | Bio-Rad Laboratories | 500-0006 | |
| Protein assay standard II | Bio-Rad Laboratories | 500-0007 | |
| Test tube 16×100 | VWR | 47729-576 | |
| Test tube 12×75 | Ultident | 170-14100005B | |
| Cell incubator | Mandel | Heracell 150 | |
| Low speed centrifuge | IEC | Centra8R | |
| High speed centrifuge | Beckman | Avanti J8 | |
| High speed rotor for 250ml bottle | Beckman | JLA16.250 | |
| High speed rotor for 50ml tube | Beckman | JA30.50 | |
| Fow cytometry | BD Bio-sciences | FACS Calibur | |
| Spectrophotometer | Beckman | Series 600 | |
| Bronchial tissue explants and sections | |||
| C57BL/6 mice (5-7 weeks old) | Charles River Laboratories, Inc. | ||
| Mouse Airway PrimaCell™ System: | CHI Scientific, Inc. | 2-82001 | |
| Rib-Back Carbon Steel Scalpel Blades | Becton Dickinson AcuteCare | 371310 | #10 |
| Scalpel Handle | Fine Science Tools Inc. | 10003-12 | #7 |
| phase-contrast inverted microscope | Olympus Optical CO., LTD. | CK2 | |
| high O2 gas mixture | VitalAire Canada Inc. | ||
| modular incubator chamber | Billups-Rothenberg Inc. | MIC-101 | |
| MaxQ 4000 incubated orbital shaker | Barnstead Lab-Line, | SHKA4000-7 | |
| 12-well tissue culture plate | Becton Dickinson and Company | 353043 | |
| Plastic tissue culture dishes (100 mm) | Sarstedt, Inc. | 83.1802 | |
| Surgical scissors | Fine Science Tools Inc. | 14060-09 | Straight, sharp, 9cm longth |
| Half-curved Graefe forceps | Fine Science Tools Inc. | 11052-10 | |
| humidified CO2 incubator | Mandel Scientific Company Inc. | SVH-51023421 | |
| Histopathological examination | |||
| formalin formaldehyde | Sigma-Aldrich, Inc. | HT5011 | |
| paraffin | Fisher scientific International, Inc. | T555 | |
| ethyl alcohol | Merck KGaA, Darmstadt | EX0278-1 | |
| glutaraldehyde | Sigma-Aldrich, Inc. | G6403 | |
| Cacodylate | Sigma-Aldrich, Inc. | 31533 | |
| microscope slides | VWR Scientific Inc. | 48300-025 | 25x75mm |
| Xylene | Fisher scientific International, Inc. | X5-4 | |
| Mayer's hematoxylin | Sigma-Aldrich, Inc. | MHS16 | Funnel with filter paper |
| HCl | Fisher scientific International, Inc. | A144s-500 | |
| eosin | Sigma-Aldrich, Inc. | HT110116 | Funnel with filter paper |
| Permount™ Mounting Medium | Thermo Fisher Scientific Inc. | SP15-100 | |
| glass coverslip | surgipath medical industries, Inc. | 84503 | 24×24 #1 |
| TUNEL detection kit | In Situ Cell Death Detection, POD | 11 684 817 910 | |
| oven | Despatch Industries Inc. | LEB-1-20 | |
| rotary Microtome | Leica Microsystems Inc. | RM2145 | |
| filter paper | Whatman International Ltd. | 1003150 | #3 |
| Microscope | Nikon Imaging Japan Inc. | E800 | |
| staining dish complete | Wheaton Industries, Inc. | 900200 | including dish, rack, cover |
| 1.5 ml eppendorf tube | Sarstedt Inc. | 72.69 | 39x10mm |
| Orbital and Reciprocating Water Bath | ExpotechUSA | ORS200 | |
| phosphate buffered saline | GIBCO | 14190-144 | |
| fume hood | Nicram RD Service | 3707E |
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