Generatie Lymfatische micropartikels en Detectie van hun proapoptotische Effect op epitheelcellen
Summary
Celhuid werpen microdeeltjes (MPs) actief biologische blaasjes die kunnen worden geïsoleerd en de pathofysiologische effecten onderzocht in verschillende modellen. Hier beschrijven we een werkwijze voor het genereren MPs afgeleid van T-lymfocyten (LMPs) en voor het aantonen van hun pro-apoptotische effect op epitheelcellen.
Abstract
De belangstelling voor de biologische rol van cel-membraan afgeleide blaasjes in cel-cel communicatie is de afgelopen jaren toegenomen. Microdeeltjes (MPs) zijn een dergelijk type van vesicles, variërend in diameter van 0,1 urn tot 1 urn, en kenmerkend schuur van het plasmamembraan van eukaryote cellen die activering of apoptose. Hier beschrijven we de generering van T-lymfocyten afkomstige microdeeltjes (LMPs) van apoptotische CEM T-cellen gestimuleerd met actinomycine D. LMPs worden geïsoleerd door een meerstaps proces differentiële centrifugatie en gekarakteriseerd met behulp van flowcytometrie. Het protocol stelt ook een in situ celdood detectiemethode voor aantonen van de pro-apoptotische effect van LMPs op bronchiale epitheelcellen afkomstig uit muizen primaire luchtwegen bronchiale weefselexplantaten. Hierin beschreven werkwijzen verschaffen een reproduceerbare werkwijze voor het isoleren overvloedige hoeveelheden LMPs van apoptotische lymfocyten in vitro. LMPs afgeleidop deze wijze kan worden gebruikt om de kenmerken van diverse ziektemodellen evalueren en farmacologie en toxicologie getest. Aangezien het luchtwegepitheel biedt een beschermende fysieke en functionele barrière tussen de buitenomgeving en het onderliggende weefsel, gebruik van bronchiale weefselexplantaten plaats geïmmortaliseerde epitheliale cellijnen een effectief model voor het onderzoek ter luchtwegen luchtwegweefsel.
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
Kamerleden zijn actieve bemiddelaars van intercellulaire overspraak en hun studie is veelbelovend in veel gebieden van de wetenschap. 11 Deze studie presenteerde een gedetailleerd protocol voor in vitro grootschalige opwekking van LMPs afgeleid van een apoptotische T-cellijn. Deze Kamerleden drukken een groot repertoire van lymfocyten moleculen en zijn biologisch betrokken bij de regulatie van de cellulaire en weefselhomeostase. Echter, LMPs afkomstig van verschillende bronnen kunnen biologisch versc…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dit werk wordt ondersteund door subsidies van de Canadese Institutes of Health Research (178.918), Fonds de recherche en santé du Québec – Vision Health Research Network.
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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