Name: induction of graft-versus-host disease and in vivo t cell monitoring using an mhc-matched murine model
Uploaded: 2012-08-29T12:00:00
Description: Watch this Scientific Journal Video about Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model at JoVE.com

Vi står i gæld til Alice Gaughan og Jiao-Jing Wang hvis fremragende teknisk support, intellektuelle input, og moralsk støtte var medvirkende til at flytte disse undersøgelser frem. Disse undersøgelser blev støttet af NIH tilskud AI036532 til Gah.

1. Letal bestråling <ol><li> Placere op til 10 recipientmus i en microisolator bur forenelig med strålerøret, der skal anvendes. </li><li> Bestråle i 2 lige store doser opsummering total dosis (totaldosis = 9 cGy for BALB.B modtagere). Second bestråling skal være 3 timer efter første. Injektion bør være mellem 4-6 timer efter den endelige bestråling. Bestråle mus i enten Cs 137 kilde eller RS2, 000, strålerøret. </li><li> Efter den anden strålingsdosis, gemme mus i microisolator bur med syrnet...

<ol>
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Protokollen til induktion GVHD i mus præsenteret her repræsenterer en klinisk relevant model af murin GVHD. Oprindeligt oprettet af Berger et al. I 1994, er det C57BL / 6 i BALB.B stamme kombination MHC-matchet, med GVHD dødelighed medieret af CD4 afhængige, CD8 T effektorer 2, meget ligner den mest almindelige kliniske scenarie 3. Det er kendt, at transplantation af CD8 T-celler alene ikke forårsager GVHD i denne model, men sygdomsprogression er betydeligt værre, når både CD4-og C...

<table border="1"><tbody><tr><td> Navn reagens </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> RPMI 1640 </td><td> Invitrogen </td><td> 12633-012 </td><td></td></tr><tr><td> Føtalt kalveserum </td><td> Invitrogen </td><td> 10439016 </td><td></td></tr><tr><td> 40 uM Cell Strainer </td><td> BD Biosciences </td><td> 352.340 </td><td></td></tr><tr><td> CD3e-Biotin </td><td> Miltenyi Biotech </td><td> 130-093-021 </td><td></td></tr><tr><td> Anti-biotin Microbeads </td><td> Miltenyi Biotech </td><td> 130-091-147 </td><td></td></tr><tr><td> CD8a Mikroperler </td><td> Miltenyi Biotech </td><td> 130-049-401 </td><td> Anvendes til at udtømme CD8 T-celler fra milt. </td></tr><tr><td> CD8a Oprensning Antibody Cocktail </td><td> Miltenyi Biotech </td><td> 130-095-236 </td><td> Anvendes til oprensningCD8 T-celler fra milt. </td></tr><tr><td> D-luciferin </td><td> Caliper Life Sciences </td><td> 122.796 </td><td></td></tr></tbody></table>

induction of graft-versus-host disease and in vivo t cell monitoring using an mhc-matched murine model

Graft-versus-vært sygdom (GVHD) er den begrænsende barriere for en bred anvendelse af knoglemarvstransplantation som en kurativ terapi for en række hæmatologiske mangler. GVHD er forårsaget af modne alloreaktive T-celler til stede i knoglemarven graft, der infunderes i modtageren og beskadige vært organer. Men i mus, skal T-celler sættes til knoglemarven inoculum at forårsage GVHD. Selv om omfattende arbejde er blevet udført for at karakterisere T-celle responser efter transplantation, bioluminescerende billedteknologi er en ikke-invasiv fremgangsmåde til overvågning af T-celle trafficking mønstre in vivo. Efter letal bestråling, er recipientmus transplanteret med knoglemarvsceller og splenocytter fra donormus. T-celledelmængder fra L2G85.B6 (transgene mus, der konstitutivt udtrykker luciferase) er inkluderet i transplantatet. Ved kun at transplantere visse T-celledelmængder er man i stand til at spore specifikke T-celledelmængder in vivo,og ud fra det sted, udvikler hypoteser om betydningen af ​​specifikke T-celledelmængder at fremme GVHD på forskellige tidspunkter. Ved forudbestemte intervaller efter transplantation, der recipientmus filmede med en Xenogen IVIS CCD kamera. Lysintensiteten kan kvantificeres ved hjælp af Living Billede software til at generere et pseudo-farvebillede baseret på foton intensitet (rød = høj intensitet, violet = lav intensitet). Mellem 4-7 dage efter transplantationen, begynder recipientmus at vise kliniske tegn på GVHD. Cooke et al. En udviklet et scoringssystem til kvantificering af sygdomsprogression baseret på recipientmus pels tekstur, hudens integritet, aktivitet, vægttab, og kropsholdning. Mus er scoret dagligt, og aflivet når de bliver døende. Recipientmus generelt blevet døende 20-30 dage efter transplantationen. Murine modeller er værdifulde værktøjer for at studere immunologi GVHD. Selektivt transplantere især T-celle delmængder alnedture til omhyggelig identifikation af de roller hver delmængde spiller. Ikke-invasiv sporing T-cellereaktioner in vivo tilføjer endnu et lag af værdi for murine GVHD model.

Watch this Scientific Journal Video about Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model at JoVE.com

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murin knoglemarvstransplantation er en meget anvendt teknik til at undersøge immunologiske mekanismer for transplantat-versus-host-sygdom hos mennesker. Muligheden for at overvåge T-celle trafficking mønstre<em&gt; In vivo</em&gt; Giver mulighed for detaljeret analyse af udviklingen og bevarelsen af ​​T-celle responser under graft-versus-host sygdom.

Induktion af graft-versus-host sygdom og<em&gt; In Vivo</em&gt; T Cell Monitoring Brug en MHC-matchet murin model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

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Immunology and Infection

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

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