JoVE Journal > Biology
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
생물학

Intestinal epitelregenerering som reaktion på ioniserende bestråling

Published: July 27, 2022
doi:
10.3791/64028
, , , ,
1Department of Medicine,Renaissance School of Medicine at Stony Brook University, 2Department of Physiology and Biophysics,Renaissance School of Medicine at Stony Brook University

Summary

Mave-tarmkanalen er et af de mest følsomme organer for skade ved stråleterapeutiske kræftbehandlinger. Det er samtidig et organsystem med en af de højeste regenerative kapaciteter efter sådanne fornærmelser. Den præsenterede protokol beskriver en effektiv metode til at studere tarmepithelets regenerative kapacitet.

Abstract

Tarmepitelet består af et enkelt lag af celler, men indeholder flere typer terminalt differentierede celler, som genereres ved aktiv spredning af intestinale stamceller placeret i bunden af tarmkrypter. Under tilfælde af akut tarmskade gennemgår disse aktive tarmstamceller imidlertid celledød. Gammabestråling er en meget anvendt kolorektal cancerbehandling, som, selvom den er terapeutisk effektiv, har den bivirkning, at den nedbryder den aktive stamcellepulje. Faktisk oplever patienter ofte gastrointestinalt strålingssyndrom, mens de gennemgår strålebehandling, delvis på grund af aktiv stamcelleudtømning. Tabet af aktive tarmstamceller i tarmkrypter aktiverer en pulje af typisk hvilende reserve intestinale stamceller og inducerer dedifferentiering af sekretoriske og enterocytprecursorceller. Hvis ikke for disse celler, ville tarmepitelet mangle evnen til at komme sig efter strålebehandling og andre sådanne store vævsfornærmelser. Nye fremskridt inden for afstamningssporingsteknologier muliggør sporing af aktivering, differentiering og migration af celler under regenerering og er med succes blevet anvendt til at studere dette i tarmen. Denne undersøgelse har til formål at skildre en metode til analyse af celler i musens tarmepitel efter strålingsskade.

Introduction

Det menneskelige tarmepitel ville dække omtrent overfladen af en halv badmintonbane, hvis den blev placeret helt fladt1. I stedet komprimeres dette enkelt cellelag, der adskiller mennesker fra indholdet af deres tarme, til en række fingerlignende fremspring, villi og fordybninger, krypter, der maksimerer tarmens overfladeareal. Cellerne i epitelet differentierer langs en krypt-villusakse. Villus består primært af næringsstofabsorberende enterocytter, slimudskillende bægerceller og de hormonproducerende enteroendokrine celler, mens krypterne primært består af defensinproducerende Paneth-celler, aktive og reservestamceller og stamceller 2,3,4,5. Desuden genererer den tovejskommunikation, disse celler har med stromale og immunceller i det underliggende mesenkymale rum og lumens mikrobiota, et komplekst netværk af interaktioner, der opretholder tarmhomeostase og er afgørende for genopretning efter skade 6,7,8.

Tarmepitelet er det hurtigst selvfornyende væv i menneskekroppen med en omsætningshastighed på 2-6 dage 9,10,11. Under homeostase deler aktive stamceller i bunden af tarmkrypter (kryptbasesøjleceller), der er kendetegnet ved ekspressionen af leucinrige gentagne holdige G-proteinkoblede receptor 5 (LGR5), hurtigt og tilvejebringer stamceller, der adskiller sig i alle andre intestinale epitellinjer. På grund af deres høje mitotiske hastighed er aktive stamceller og deres umiddelbare forfædre imidlertid særligt følsomme over for gammastrålingsskader og gennemgår apoptose efter bestråling 5,12,13,14. Efter deres tab gennemgår reservestamceller og ikke-stamceller (subpopulation af forfædre og nogle terminalt differentierede celler) i tarmkrypter aktivering og genopfylder basalkryptrummet, som derefter kan rekonstituere cellepopulationer af villi og dermed regenerere tarmepitelet15. Ved hjælp af afstamningssporingsteknikker har flere forskergrupper vist, at reservestamceller (hvilende) er i stand til at understøtte regenerering ved tab af aktive stamceller 13,16,17,18,19,20,21,22. Disse celler er karakteriseret ved tilstedeværelsen af polycomb kompleks protein 1 onkogen (Bmi1), mus telomerase revers transkriptase gen (mTert), Hop homeobox (Hopx) og leucin-rige gentage protein 1 gen (Lrig1). Derudover har det vist sig, at ikke-stamceller er i stand til at genopbygge tarmkrypter ved skade 23,24,25,26,27,28,29,30,31. Det har især vist sig, at forfædre til sekretoriske celler og enterocytter gennemgår dedifferentiering ved skade, vender tilbage til stamlignende celler og understøtter regenerering af tarmepitelet. Nylige undersøgelser har identificeret celler, der udtrykker flere markører, der har kapacitet til at erhverve stammelignende egenskaber ved skade (såsom DLL+, ATOH1+, PROX1+, MIST1+, DCLK1+)32,33,34,35,36. Overraskende viste Yu et al., at selv modne Paneth-celler (LYZ +) kan bidrage til intestinal regenerering37. Ud over at forårsage apoptose af intestinale epitelceller og forstyrre epitelbarrierefunktionen resulterer bestråling desuden i dysbiose af tarmfloraen, immuncelleaktivering og initiering af et proinflammatorisk respons samt aktivering af mesenkymale og stromale celler38,39.

Gammastråling er et værdifuldt terapeutisk værktøj i kræftbehandling, især for kolorektal tumorer40. Bestråling påvirker imidlertid signifikant intestinal homeostase ved at fremkalde skade på cellerne, hvilket fører til apoptose. Strålingseksponering forårsager flere forstyrrelser, der bremser patientens opsving og er præget af slimhindeskade og betændelse i den akutte fase og diarré, inkontinens, blødning og mavesmerter på lang sigt. Denne panoply af manifestationer kaldes gastrointestinal strålingstoksicitet. Derudover kan strålingsinduceret progression af transmural fibrose og/eller vaskulær sklerose først manifestere sig år efter behandlingen38,41. Samtidig med selve skaden inducerer stråling et reparationsrespons i tarmceller, der aktiverer signalveje, der er ansvarlige for at initiere og orkestrere regenerering42. Strålingsinduceret tyndtarmssygdom kan stamme fra bækken- eller abdominal strålebehandling, der leveres til andre organer (såsom livmoderhalsen, prostata, bugspytkirtel, endetarm)41,43,44,45,46. Tarmbestrålingsskader er således et væsentligt klinisk problem, og en bedre forståelse af den resulterende patofysiologi vil sandsynligvis fremme udviklingen af interventioner til lindring af gastrointestinale komplikationer forbundet med strålebehandling. Der er andre teknikker, der gør det muligt at undersøge det regenerative formål med tarmepitelet bortset fra stråling. Transgene og kemiske murinmodeller til undersøgelse af inflammation og regenerering derefter er blevet udviklet47. Dextran natriumsulfat (DSS) inducerer betændelse i tarmen og fører til udvikling af egenskaber svarende til inflammatoriske tarmsygdomme48. En kombination af DSS-behandling med den pro-kræftfremkaldende forbindelse azoxymethan (AOM) kan resultere i udvikling af colitis-associeret cancer48,49. Iskæmi reperfusionsinduceret skade er en anden metode, der anvendes til at studere det regenerative potentiale i tarmepitelet. Denne teknik kræver erfaring og kirurgisk viden50. Desuden forårsager ovennævnte teknikker andre typer skader end stråling og kan føre til involvering af forskellige regenereringsmekanismer. Derudover er disse modeller tidskrævende, mens strålingsteknikken er ret kort. For nylig er in vitro-metoder, der anvender enteroider og colonoider genereret fra tarm og tyktarm, blevet anvendt i kombination med strålingsskade til at studere mekanismerne for intestinal regenerering51,52. Disse teknikker rekapitulerer imidlertid ikke fuldt ud det organ, de modellerer53,54.

Den fremlagte protokol indeholder beskrivelsen af en murinmodel af gammastrålingsskade i kombination med en genetisk model, der efter tamoxifenbehandling muliggør sporing af slægter, der stammer fra reservestamcellepopulationen (Bmi1-CreER; Rosa26eYFP). Denne model anvender en 12 Gy totalkropsbestråling, som inducerer signifikant nok tarmskade til at aktivere reservestamceller, mens den stadig giver mulighed for efterfølgende undersøgelse af tarmregenerativ kapacitet inden for 7 dage efter skade55.

Protocol

Alle mus blev anbragt i Division of Laboratory Animal Resources (DLAR) ved Stony Brook University. Stony Brook University Institutional Animal Care and Use Committee (IACUC) godkendte alle undersøgelser og procedurer, der involverede dyreforsøgspersoner. Forsøg med dyr blev udført i nøje overensstemmelse med den godkendte dyrehåndteringsprotokol (IACUC # 245094). BEMÆRK: Musstammer B6;129-Bmi1 tm1(cre/ERT)Mrc/J (Bmi1-Cre ER) og B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J (<…

Representative Results

Anvendelsen af 12 Gy totalkropsbestråling (TBI) i kombination med sporing af murin genetisk afstamning muliggør en grundig analyse af konsekvenserne af strålingsskader i tarmen. For at starte, Bmi1-CreER; Rosa26eYFP-mus modtog en enkelt tamoxifeninjektion, som inducerer forbedret gult fluorescerende protein (EYFP) ekspression inden for en Bmi1+ reservestamcellepopulation. To dage efter tamoxifeninjektionen gennemgik musene bestråling eller skinbestråling. Tre timer før euta…

Discussion

Denne protokol beskriver en robust og reproducerbar strålingsskademodel. Det giver mulighed for præcis analyse af ændringerne i tarmepitelet i løbet af 7 dage efter skaden. Det er vigtigt, at de valgte tidspunkter afspejler afgørende stadier af skade og er kendetegnet ved tydelige ændringer i tarmen (skade, apoptose, regenerering og normaliseringsfaser)60. Denne bestrålingsmodel er blevet fastlagt og omhyggeligt vurderet, hvilket viser en passende manifestation af efterligningsskade som den…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Forfatterne ønsker at anerkende Stony Brook Cancer Center Histology Research Core for eksperthjælp med forberedelse af vævsprøver og Division of Laboratory Animal Resources ved Stony Brook University for hjælp til dyrepleje og håndtering. Dette arbejde blev støttet af tilskud fra National Institutes of Health DK124342 tildelt Agnieszka B. Bialkowska og DK052230 til Dr. Vincent W. Yang.

Materials

1 mL syringe BD 309659 –
16G Reusable Small Animal Feeding Needles: Straight VWR 20068-630 –
27G x 1/2" needle BD 305109 –
28G x 1/2" Monoject 1mL insulin syringe Covidien 1188128012 –
5-Ethynyl-2′-deoxyuridine (EdU) Santa Cruz Biotechnology sc284628A 10 mg/mL in sterile DMSO:water (1:4 v/v), aliquot and store in -20°C
Azer Scientific 10% Neutral Buffered Formalin Fisher Scientific 22-026-213 –
B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J The Jackson Laboratory Strain #:006148
B6;129-Bmi1tm1(cre/ERT)Mrc/J The Jackson Laboratory Strain #:010531
Bovine Serum Albumin Fraction V, heat shock Millipore-Sigma 3116956001
Chicken anti-GFP Aves GFP-1020
Click-IT plus EdU Alexa Fluor 555 imaging kit, Invitrogen Thermo Fisher Scientific C10638 –
Corn oil Millipore-Sigma C8267 –
Decloaking Chamber Biocare Medical DC2012 –
Dimethyl sulfoxide (DMSO) Fisher BioReagents BP231-100 light sensitive
DNase-free proteinase K Invitrogen C10618H diluted 25x in DPBS
Donkey anti-chicken AF647 Jackson ImmunoResearch 703-605-155
DPBS Fisher Scientific 21-031-CV –
Eosin Fisher Scientific S176
Fluorescence Microscope Nikon Eclipse 90i Bright and fluoerescent light, with objectives: 10X, 20X Nikon
Fluoromount Aqueous Mounting Medium Millipore-Sigma F4680-25ML
Gamma Cell 40 Exactor Best Theratronics Ltd. – 0.759 Gy min-1
Goat anti-rabbit AF488 Jackson ImmunoResearch 111-545-144
Hematoxylin Solution, Gill No. 3 Millipore-Sigma GHS332
HM 325 Rotary Microtome from Thermo Scientific Fisher Scientific 23-900-668
Hoechst 33258, Pentahydrate (bis-Benzimide) Thermo Fisher Scientific H3569 dilution 1:1000
Hydrogen Peroxide Solution, ACS, 29-32%, Spectrum Chemical Fisher Scientific 18-603-252 –
In Situ Cell Death Detection Kit, Fluorescein (Roche) Millipore-Sigma 11684795910
Liquid Blocker Super PAP PEN, Mini Fisher Scientific DAI-PAP-S-M
Lithium Carbonate (Powder/Certified ACS), Fisher Chemical Fisher Scientific L119-500 0.5g/1L dH2O
Luer-Lok Syringe sterile, single use, 10 mL VWR 89215-218 –
Methanol VWR BDH1135-4LP
Pharmco Products Ethyl alcohol, 200 PROOF Fisher Scientific NC1675398 –
Pharmco-Aaper 281000ACSCSLT Acetic Acid ACS Grade Capitol Scientific AAP-281000ACSCSLT –
Rabbit anti-Ki67 BioCare Medical CRM325
Richard-Allan Scientific Cytoseal XYL Mounting Medium Fisher Scientific 22-050-262
Scientific Industries Incubator-Genie for baking slides at 65 degree Fisher Scientific 50-728-103
Sodium Citrate Dihydrate Fisher Scientific S279-500
Stainless Steel Dissecting Kit VWR 25640-002
Superfrost Plus micro slides [size: 25 x 75 x 1 mm] VWR  48311-703
Tamoxifen Millipore-Sigma T5648 30 mg/mL in sterile corn oil, preferably fresh or short-sterm storage in -20°C, light sensitive
Tissue-Tek 24-Slide Holders with Detachable Handle Sakura 4465
Tissue-Tek Accu-Edge Low Profile Blades Sakura 4689
Tissue-Tek Manual Slide Staining Set Sakura 4451
Tissue-Tek Staining Dish, Green with Lid Sakura 4456
Tissue-Tek Staining Dish, White with Lid Sakura 4457
Tween 20 Millipore-Sigma P7949
Unisette Processing Cassettes VWR 87002-292 –
VWR Micro Cover Glasses VWR 48393-081
Xylene Fisher Scientific X5P-1GAL
DOWNLOAD MATERIALS LIST

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Keywords: Intestinal Epithelial RegenerationIonizing RadiationGamma IrradiationColorectal CancerLineage TracingTamoxifenEdUBouin’s FixativeGamma IrradiatorCre-mediated Recombination
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Orzechowska-Licari, E. J., LaComb, J. F., Giarrizzo, M., Yang, V. W., Bialkowska, A. B. Intestinal Epithelial Regeneration in Response to Ionizing Irradiation. J. Vis. Exp. (185), e64028, doi:10.3791/64028 (2022).
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