Generation af lymfatisk mikropartikler og påvisning af deres proapoptotiske Påvirkning af luftvejsepitelceller
Summary
Cellemembran-kaste mikropartikler (MPS) er aktive biologiske vesikler, som kan isoleres, og deres patofysiologiske virkninger undersøgt i forskellige modeller. Her beskriver vi en fremgangsmåde til generering af MP'er afledt fra T-lymfocytter (LMPs) og til at demonstrere deres proapoptotiske virkning på luftveje epitelceller.
Abstract
Interessen for de biologiske roller cellemembran-afledte vesikler i celle-celle kommunikation er steget i de senere år. Mikropartikler (MPS) er en sådan type af vesikler, en diameter på fra 0,1 um til 1 um, og typisk kaste fra plasmamembranen af eukaryote celler, der undergår aktivering eller apoptose. Her beskriver vi genereringen af T lymfocyt-afledt mikropartikler (LMPs) fra apoptotiske CEM T-celler stimuleret med actinomycin D. LMPs isoleres gennem en flertrins differentialcentrifugering proces og karakteriseret ved anvendelse af flowcytometri. Denne protokol viser også en in situ celledød påvisningsmetode for at demonstrere proapoptotiske virkning LMPs på bronchieepithelceller celler afledt fra muse primær respiratorisk bronchiale vævseksplantater. Heri beskrevne fremgangsmåder giver en reproducerbar procedure til isolering af rigelige mængder LMPs fra apoptotiske lymfocytter in vitro. LMPs afledtpå denne måde kan anvendes til at vurdere karakteristika for forskellige sygdomsmodeller og til farmakologi og toksikologi test. Da luftvejsepitel tilbyder en beskyttende fysisk og funktionel barriere mellem det eksterne miljø og det underliggende væv, anvendelse af bronkial vævseksplantater snarere end immortaliserede epiteliale cellelinjer tilvejebringer en effektiv model for undersøgelser, der kræver luftvejene tarmkanalen væv.
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
Parlamentsmedlemmer er aktive mediatorer af intercellulære krydstale og deres undersøgelse er lovende på mange områder af videnskaben. 11 Denne undersøgelse præsenteret en detaljeret protokol for in vitro i stor målestok af LMPs afledt af en apoptotisk T-cellelinie. Disse parlamentsmedlemmer udtryk et stort repertoire af lymfocyt-molekyler og er biologisk impliceret i reguleringen af cellulære og vævshomeostase. Dog kan LMPs hidrører fra forskellige kilder være biologisk anderledes. …
Divulgations
The authors have nothing to disclose.
Acknowledgements
Dette arbejde er støttet af tilskud fra de canadiske 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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