Generasjon av lymfatisk Mikropartikler og Påvisning av deres proapoptotiske Effekt på Airway Epitelceller
Summary
Cellemembranen-skur mikropartikler (MPS) er aktive biologiske vesikler som kan isoleres og deres patofysiologiske effekter undersøkt i ulike modeller. Her beskriver vi en fremgangsmåte for å generere MP'er avledet fra T-lymfocytter (LMPs), og for å demonstrere deres proapoptotiske effekt på luftveis epitelceller.
Abstract
Interessen for de biologiske roller cellemembranen-avledet vesikler i celle-celle kommunikasjon har økt de siste årene. Mikropartikler (MPS) er en slik type vesikler, som varierer i diameter fra 0,1 pm til 1 pm, og vanligvis skur fra plasmamembranen av eukaryote celler som gjennomgår apoptose eller aktivering. Her beskriver vi generering av T-lymfocytt-avledede mikropartikler (LMPs) fra apoptotiske CEM T-celler stimulert med actinomycin D. LMPs isoleres gjennom en flertrinnsprosess differensialsentrifugering og karakterisert ved hjelp av flow cytometri. Denne protokollen presenterer også en in situ celledød deteksjonsmetode for å demonstrere apoptosiske effekten av LMPs på bronkial epitelceller som stammer fra mus primære luftveis bronkial vev explants. Fremgangsmåtene beskrevet heri tilveiebringe en reproduserbar fremgangsmåte for isolering av rikelig mengder LMPs fra apoptotiske lymfocytter in vitro. LMPs avledetpå denne måte kan brukes til å evaluere egenskapene til forskjellige sykdomsmodeller, og for farmakologi og toksikologi testing. Bruken av bronkial vev eksplantater fremfor immortaliserte epiteliale cellelinjer gitt at luftveisepitel har en beskyttende fysiske og funksjonelle barriere mellom det ytre miljø og det underliggende vev, gir en effektiv modell for undersøkelser som krever luftveiskanalen vev.
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 formidlere av interkrysstale og deres studie er lovende i mange områder av vitenskap. 11 Denne studien presentert en detaljert protokoll for in vitro storskala generasjon LMPs avledet fra en apoptotisk T cellelinje. Disse parlamentsmedlemmer uttrykker et stort repertoar av lymfocytt molekyler og er biologisk innblandet i reguleringen av mobilnettet og vev homeostase. Imidlertid kan LMPs avledet fra forskjellige kilder være biologisk forskjellig. 4,9,12,13 </…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbeidet er støttet med tilskudd fra den kanadiske Institutes of Health Research (178 918), Fonds de recherche no 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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