JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Immunology and Infection

Analyse af mastcellernes funktioner i Vivo Ved hjælp af 'Mast Cell Knock-in' Mus

Published: May 27, 2015
doi:
10.3791/52753
, , , , ,
1Department of Pathology,Stanford University School of Medicine, 2Department of Microbiology & Immunology,Stanford University School of Medicine

Summary

Vi beskriver en metode til generering af in vitro-afledte mastceller, deres engraftment i mastcelle-mangelfulde mus og analyse af fænotypen, antal og fordeling af engrafterede mastceller på forskellige anatomiske steder. Denne protokol kan bruges til at vurdere mastcellernes funktioner in vivo.

Abstract

Mastceller (MCs) er hæmatopoietiske celler, der findes i forskellige væv, og er især rigelige på steder, der udsættes for det ydre miljø, såsom hud, luftveje og mave-tarmkanalen. Bedst kendt for deres skadelige rolle i IgE-afhængige allergiske reaktioner, har MCs også vist sig som vigtige aktører i værtsforsvaret mod gift og invaderende bakterier og parasitter. MC-fænotype og -funktion kan påvirkes af mikromiljøfaktorer, der kan variere afhængigt af anatomisk placering og/eller baseret på typen eller udviklingsstadiet for immunrespons. Af denne grund har vi og andre begunstiget in vivo tilgange over in vitro metoder til at få indsigt i MC funktioner. Her beskriver vi metoder til generering af mus knoglemarvs-afledte dyrkede MC’er (BMCMCs), deres adoptivoverførsel til genetisk MC-mangelfuld mus, og analysen af antallet og fordelingen af adoptiv-overførte MC’er på forskellige anatomiske steder. Denne metode, der kaldes“mast celle knock-in”-metoden, er blevet anvendt i vid udstrækning i løbet af de sidste 30 år til at vurdere MCs og MC-afledte produkter in vivo. Vi diskuterer fordele og begrænsninger ved denne metode i lyset af alternative tilgange, der er blevet udviklet i de senere år.

Introduction

Mastceller (MCs) er hæmatopoietiske celler, der opstår fra pluripotente knoglemarvs forfædre1-3. Efter knoglemarvsudstridelse migrerer MCs-forfædre ind i forskellige væv, hvor de udvikler sig til modne MCs under indflydelse af lokale vækstfaktorer1-3. Vævsbeboende MCs er strategisk placeret på værtsmiljøgrænseflader, såsom huden, luftvejene og mave-tarmkanalen, hvor de opfører sig som en første forsvarslinje mod eksterne fornærmelser3-6. MCs er ofte subklassificeret baseret på deres “baseline” fænotypiske egenskaber og deres anatomiske placeringer. Hos mus er der beskrevet to typer MC’er: “bindevævstype” MCs (CTMCs) og slimhinde-MCs (MMCs)1-3,7,8. CTMCs er ofte placeret omkring venules og nær nervefibre, og bor i serosale hulrum, mens MMCs indtager intraepithelial steder i tarmen og respiratorisk slimhinde1-3.

Talrige metoder er blevet anvendt til at studere biologiske funktioner af MCs9-13. Mange grupper har fokuseret på in vitro tilgange ved hjælp af enten cellelinjer (såsom den menneskelige MC linjer HMC114 eller LAD215,16), in vitro afledte MC (såsom menneskelige perifere blod-afledte MC17, eller mus knoglemarv-afledt kultur18, føtal hudafledte dyrkede MC’er [FSCMCs]19 og peritoneale cellebaserede MCs [PCMCs]20) eller ex vivo isolerede MCS fra forskellige anatomiske steder. Alle disse modeller er meget udbredt til at studere molekylære detaljer i MC biologi, såsom signalering veje involveret i MC aktivering. Et vigtigt aspekt af MCs biologi er imidlertid, at deres fænotypiske og funktionelle egenskaber(f.eks.cytoplasmisk granulatproteaseindhold eller reaktion på forskellige stimuli) kan moduleres ved anatomisk placering og mikromiljø2,7. Da den nøjagtige blanding af sådanne faktorer, der opstår in vivo, kan være vanskelig at reproducere in vitro, går vi ind for at bruge in vivo-tilgange til at få indsigt i MCs-funktioner9.

Der findes flere musestammer med genetisk MC-mangel, såsom de udbredte WBB6F1–Kit W/W-v eller C57BL/6-Kit W-sh/W-sh mus. Disse mus mangler udtryk og/eller aktivitet af KIT (CD117), receptoren for den vigtigste MC vækstfaktor stamcellefaktor (SCF)21,22. Som følge heraf har disse mus en dyb MC-mangel, men har også yderligere fænotypiske abnormiteter relateret til deres c- kit-mutationer (i WBB6F1–Kit W/ W-v mus) eller til virkningerne af den store kromosomale inversion, der resulterer i reduceret c-kit-udtryk (i C57BL/6-Kit W-sh/W-sh mus) 9,10,12,23. For nylig er flere stammer af mus med c-kit-uafhængigkonstituerende MC-mangel blevet rapporteret24-26. Alle disse mus og nogle yderligere nye typer af inducible MC-mangelfulde mus er for nylig blevet gennemgået i detaljer9,10,13.

Her beskriver vi metoder til generering af mus knoglemarv-afledte dyrkede MC’er (BMCMCs), deres adoptivoverførsel til MC-mangelfuld mus, og analysen af antallet og fordelingen af adoptiv-overførte MC’er på forskellige anatomiske steder. Denne såkaldte “mastcelle knock-in”-metode kan bruges til at vurdere MCs og MC-afledte produkter in vivo. Vi diskuterer fordele og begrænsninger ved denne metode i lyset af alternative tilgange, der er blevet udviklet i de senere år.

Protocol

Al dyrepleje og -forsøg blev udført i overensstemmelse med retningslinjerne fra National Institutes of Health og med den specifikke godkendelse af Institutional Animal Care and Use Committee fra Stanford University. 1. Generering og karakterisering af knoglemarvsbaserede kulturperler (BMCMCs). Bemærk: Donor BMCMCs bør genereres fra knoglemarvsceller med samme genetiske baggrund som modtageren MC-mangelfuld mus. Mandlige donor BMCMCs er ikke egnede til engraftment …

Representative Results

En oversigt over metoden ‘mastcelle knock-in’ er vist i figur 1og omfatter generering af BMCMCs, antallet af celler, der skal indpodes i.p., i.d. eller i.v. i MC-mangelfulde mus (antallet kan varieres, hvis det er angivet på grundlag af forsøgsdesignet) og intervallet mellem engraftment og eksperiment afhængigt af injektionsstedet (dette interval kan også variere, hvis det er angivet; f.eks.øges indholdet af lagrede mæglere i MC cytoplasmiske granulater støt med tiden38</sup…

Discussion

Næsten 30 år efter den første beskrivelse38, den ‘mast celle knock-in‘ tilgang fortsætter med at give værdifulde oplysninger om, hvad MCs kan gøre eller ikke kan gøre in vivo. De funktioner, MCs var længe menes at være begrænset til deres rolle i allergi. Data genereret ved hjælp af ‘mast celle knock-in‘ tilgang har ændret denne opfattelse, ved at fremlægge bevis for, at MCs kan blandt andre funktioner, spille kritiske roller i værtsforsvaret mod visse patogener4,3…

Disclosures

The authors have nothing to disclose.

Acknowledgements

N.G. modtager stipendier fra den franske “Fondation pour la Recherche Médicale FRM” og Philipp Foundation; R.S. støttes af Lucile Packard Foundation for Children’s Health og Stanford NIH/NCRR CTSA award number UL1 RR025744; PS støttes af et Max Kade Fellowship fra Max Kade Foundation og det østrigske videnskabsakademi og et Schroedinger Fellowship fra den østrigske videnskabsfond (FWF): J3399-B21; SJ G. anerkender støtte fra National Institutes of Health tilskud U19 AI104209, NS 080062 og fra Tobaksrelaterede Sygdomme Research Program ved University of California; L.L.R. anerkender støtte fra Arthritis National Research Foundation (ANRF) og National Institutes of Health grant K99AI110645.

Materials

1% Antibiotic-Antimycotic Solution Corning cellgro 30-004-Cl
3 ml Syringe Falcon 309656
35 mm x 10 mm Dish Corning cellgro 430588
5 ml Polystyrene Round Bottom Tube Falcon 352058
Acetic Acid Glacial Fisher Scientific A35-500
Alcian Blue 8GX Rowley Biochemical Danver 33864-99-2
Allegra 6R Centrifuge Beckman
Anti-mouse CD16/32 (clone 93) Purified eBioscience 14-0161-81
2-Mercaptoethanol Sigma Aldrich M7522
BD 1 ml TB Syringe BD Syringe 309659
BD 22G x1 (0.7 mm x 25 mm) Needles BD Precision Glide Needle 205155
BD 25G 5/8 Needles BD Syringe 305122
BD 30G x1/2 Needles BD Precision Glide 305106
Blue MAX Jr, 15 ml Polypropylene Conical Tube Falcon 352097
Chloroform Fisher Scientific C298-500
Cytoseal 60 Mounting Medium Richard-Allan Scientific 8310-4
Cytospin3 Shandon NA
DakoCytomation pen Dako S2002
Dulbecco Modified Eagle Medium (DMEM) 1x Corning cellgro 15-013-CM
Ethanol Sigma Aldrich E 7023-500ml
Fetal Bovine Serum Heat Inactivated Sigma Aldrich F4135-500ml
FITC Conjugated IgG2b K Rat Isotype Control eBioscience 14-4031-82
Fluorescein Isotiocyanate (FITC) Conjugated Anti-mouse KIT (CD117; clone 2B8) eBioscience 11-1171-82
Formaldehyde Fisher Scientific F79-500
Giemsa Stain Modified Sigma Aldrich GS-1L
Isothesia Henry Schein Animal Health 29405
May-Grunwald Stain Sigma Aldrich MG-1L
Multiwell 6 well plates Falcon 35 3046
Olympus BX60 Microscope Olympus NA
Paraplast Plus Tissue Embedding Medium Fisher Brand 23-021-400
PE Conjugated IgG Armenian Hamster Isotype Control eBioscience 12-4888-81
Phosphate-Buffered-Saline (PBS) 1x Corning cellgro 21-040-CV
Phycoerythrin (PE) Conjugated Anti-mouse FceRIa (clone MAR-1) eBioscience 12-5898-82
Propidium Iodide Staining Solution eBioscience 00-6990-50
Recombinant Mouse IL-3 Peprotech 213-13
Safranin-o Certified Sigma Aldrich S8884
Tissue culture flasks T25 25 cm2 Beckton Dickinson 353109
Tissue culture flasks T75 75 cm2 Beckton Dickinson 353110
Toluidine Blue 1 % Aqueous LabChem-Inc LC26165-2
Recombinant Mouse SCF Peprotech 250-03
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Tags

Mast CellsIn VivoKnock-in MiceBone Marrow-derived Cultured Mast CellsAdoptive TransferAnatomical DistributionHost DefenseAllergic Reactions
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Gaudenzio, N., Sibilano, R., Starkl, P., Tsai, M., Galli, S. J., Reber, L. L. Analyzing the Functions of Mast Cells In Vivo Using ‘Mast Cell Knock-in‘ Mice. J. Vis. Exp. (99), e52753, doi:10.3791/52753 (2015).
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