Udvikling af en Ethanol-induceret Fibrotiske Lever Model i Zebrafisk at studere progenitorcelle-medieret hepatocyt Regeneration
Summary
Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.
Abstract
Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.
Introduction
Trods af de bemærkelsesværdige regenereringsevne af hepatocytter 1, som er den største parenkymalt celletype af leveren, kronisk leversvigt svækker denne evne, hvilket fører til nedsat progenitorcelle (HPC) -afhængig regenerering 2.
Kronisk leverskader er hovedsageligt stammer fra alkoholmisbrug, kronisk hepatitis C virus (HCV) infektion 3 og ikke-alkoholiske fedtlever sygdom (NAFLD) 4. Det fører til en vedvarende leverfibrose, som er forbundet med akkumuleringen af ekstracellulær matrix (ECM) proteiner. Vedvarende ECM akkumulering fordrejer intakt hepatisk arkitektur ved at danne et fibrøst arvæv 5, efterfølgende resulterer i cirrhosis med høj morbiditet og mortalitet. Mange forsøg er blevet gjort for at mindske den fibrotiske reaktion hovedsageligt ved at fokusere på inhibering profibrogenic cytokiner og aktiverede myofibroblaster 6. Sidstnævnte er primært afledt af hepatiske stjerneformige celler (HSCs), de vigtigste hepatiske ikke-parenkymceller ansvarlige for leveren ardannelse 4. Ikke desto mindre, regenerative behandlinger, der stimulerer endogene cellulære kilder, herunder HPCs at regenerere hepatocytter i overværelse af vedvarende fibrogene fornærmelser afventer yderligere undersøgelser.
Mange forsøgsmodeller af hepatisk fibrose er blevet beskrevet i pattedyr. Gentagen injektion af carbontetrachlorid (CCI4) har været meget anvendt til at inducere leverfibrose i murine og rottemodeller 7. Når det kombineres med en fedtrig (HF) kost, alkohol ført til en betydelig opregulering af profibrogenic genekspression og hepatisk fibrose 8. Mens steatose (lipid ophobning) skyldes akut eksponering alkohol, det gør leveren modtagelige for mere alvorlig leverskade 9.
Den zebrafisk, Danio rerio, har vist sig som en uvurderlig hvirveldyr modelsystem til undersøgelse regenerering. Selvomandre lavere hvirveldyr som f.eks salamandre og axolotls har en bemærkelsesværdig evne til regeneration, at zebrafisk har fordele frem for andre modelsystemer i forhold til de genmanipulation og visualisering strategier er nødvendige for at manipulere potentielle regenerativ faktorer 10. Zebrafisken repræsenterer også en attraktiv hvirveldyr model til at studere alkoholisk leversygdom (ALD) ved blot at tilføje ethanol (EtOH) til deres vand. Akut EtOH udsættelse for larver og voksne zebrafisk forårsagede leversteatose 11-13. Når voksne zebrafisk fik forlænget EtOH eksponering blev collagenaflejringen observeret med opregulering af fibrose-relaterede gener 14. Imidlertid er der et behov for at udvikle modeller til at studere leverregenerering som reaktion på EtOH som fibrogene stimulus.
For nylig har vi udviklet en EtOH-induceret fibrotisk lever model i zebrafisk 15. Vi kombinerede en hepatocyt-specifik genetisk ablationssystem med EtOH behandling i larvernes og Adult zebrafisk. Vi genererede to transgene linjer, Tg (fabp10a: CFP-NTR) GT1 og Tg (fabp10a: mCherry-NTR) GT2, hvor E.coli nitroreduktase (NTR) er fusioneret til cyan og mCherry fluorescerende protein, henholdsvis under kontrol af hepatocyt-specifik fedtsyrebindende protein 10a, lever basic (fabp10a) promotor. I dette system NTR konverterer en ikke-toksisk prodrug metronidazol (MTZ) i en DNA inter-streng tværbindingsmiddel 16, inducere eksplicit død af hepatocytter. Under anvendelse af denne model påviste vi, at en population af leverceller, som reagerer på Notch signalering, omregnet til hepatocytter i nær fravær af hepatocytter og i overskud af ECM. Vi har udpeget disse celler som HPC'er. Desuden gennem kemiske skærme, vi identificeret lille molekyle aktivatorer af Wnt-signalering og hæmmere af Notch signalering, som styrker hepatocyt regenerering i fibrøse leveren. therefore, vores fibrotisk lever model i zebrafisk repræsenterer en fantastisk kemisk screening-system i forhold til celle kultur- eller pattedyr-baserede screening system. Det er et in vivo-system med betydelig omkostnings- og tidsbesparelser. Her beskriver vi de nærmere regler for oprettelse af en EtOH-induceret fibrotisk lever model og til at udføre kemiske skærme ved hjælp af denne model i zebrafisk. Desuden blev tidsforløb analyser udført for at undersøge, hvordan hepatocyt regenerering forekommer i den fibrotiske leveren. Denne protokol vil give et uvurderligt værktøj til at studere de mekanismer og strategier til at forbedre hepatocyt regenerering i fibrøse leveren.
Protocol
Representative Results
Discussion
Vi observerede HPC-medieret hepatocyt regenerering i EtOH / MTZ-behandlede genvinder lever, hvilket tyder på, at selv under tilstedeværelse af væsentlig mængde ECM-proteiner, herunder fibrillære type I collagen, de HPC'er bevarer deres kompetence til at regenerere som hepatocytter. Det MTZ kun behandling steg ikke aflejring af ECM-proteiner betydeligt, mens EtOH eneste behandling ikke inducerede HPC aktivering 15. Ved at udnytte den kombinerede EtOH / MTZ behandling, var vi i stand til at undersøge …
Divulgations
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet delvist af tilskud fra GTEC (2731336 og 1411318), NIH (K01DK081351), og NSF (1.354.837) til CHS Vi takker Alem Giorgis for kritisk læsning af manuskriptet.
Materials
| Calcium sulfate hemihydrate (CaSO4) | Acros | AC385355000 | |
| Magnesium sulfate (MgSO4) | EMD | MX0075 | |
| 1,4-Piperazinediethanesulfonic acid (PIPES) | Sigma-Aldrich | P6757 | |
| Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) | Sigma-Aldrich | E3889 | |
| Ethanol | Sigma-Aldrich | E7023 | 200 proof |
| Formaldehyde | Fisher Scientific | F79-500 | |
| Metronidazole (MTZ) | Sigma-Aldrich | M3761 | |
| 1-phenyl-2-thiourea (PTU) | Sigma-Aldrich | P7629 | |
| 3-amino benzoic acid ethyl ester (Tricaine) | Sigma-Aldrich | A5040 | |
| Phosphate-buffered saline (PBS) tablet | Amresco | E404 | Dissolve one tablet with 100 ml distilled water |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D2438 | |
| Bovine serum albumin | Fisher Scientific | BP1600 | |
| Triton X-100 | Fisher Scientific | BP151 | |
| Low-melting agarose | Amresco | BP165 | |
| Stem Cell Signaling Compound Library | Selleck Chemicals | L2100 | 10mM stock in DMSO |
| ActiProbe-1K Library | Timtec | ActiProbe-1K | 10mM stock in DMSO |
| SB 415286 | Selleck Chemicals | S2729 | Dissolve with DMSO to 10mM |
| CHIR-99021 | Selleck Chemicals | S2924 | Dissolve with DMSO to 10mM |
| Anti-Collagen I antibody | Abcam | ab23730 | Use at 1:100 for immunostaining, reacts with fish |
| AlexaFluor 647 Donkey anti-rabbit IgG (H+L) | Molecular Probes | A31573 | Use at 1:200 for immunostaining |
| Mounting media (Vectorshield) | Vector Laboratories | H-1400 | |
| 100 mm petri dish | VWR | 25384-088 | |
| 24-well plate | VWR | 10062-896 | |
| Forceps | Fine Science Tools | 11255-20 | Dumont #55 |
| Glass slide | VWR | 48312-003 | 75×25 mm |
| Cover glass | VWR | 48366-045 | 18 mm |
| Plastic wrap | Fisher Scientific | 22305654 | |
| Aluminum foil | Fisher Scientific | 1213100 | |
| Kimwipes | Kimberly-Clark | 34155 | |
| Vibrotome | Leica | VT1000 S | |
| Stereo microscope | Leica | M80 | |
| Epifluoresent microscope | Leica | M205 FA | |
| Confocol microscope | Zeiss | LSM700 |
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