Etablering af Dual humaniseret TK-NOG muse model for HIV-associeret lever patogenesen
Summary
Denne protokol giver en pålidelig metode til at etablere humaniserede mus med både menneskelige immunsystem og leverceller. Dual rekonstitueret immundefekt mus opnået via intrasplenisk injektion af humane hepatocytter og CD34+ hæmatopoietiske stamceller er modtagelige for human immuninsufficiens virus-1 infektion og rekapitulere leverskader som observeret i HIV-inficerede patienter.
Abstract
På trods af den øgede forventede levetid for patienter inficeret med humant immundefektvirus-1 (HIV-1), leversygdom er opstået som en almindelig årsag til deres sygelighed. Den lever immunopatologi forårsaget af HIV-1 forbliver undvigende. Små xenograft dyremodeller med humane hepatocytter og humant immunsystem kan rekapitulere den menneskelige biologi af sygdommens patogenese. Heri er en protokol beskrevet for at etablere en dual humaniseret musemodel gennem humane hepatocytter og CD34+ hæmatopoietiske Stem/progenitorceller (HSPCs) transplantation, at studere leveren immunopatologi som observeret hos HIV-inficerede patienter. For at opnå dobbelt rekonstitution, mand TK-NOG (NOD. CG-Prkdcscid Il2rgtm1Sug TG (Alb-tk) 7-2/shijic) mus intraperitonealt injiceres med ganciclovir (gcv) doser for at eliminere mus transgene leverceller, og med treosulfan til nonmyeloablativ konditionering, som begge fremme menneskelig hepatocyt (HEP) engraftment og menneskelige immunsystem (hans) udvikling. Humant albumin (Alb) niveauer er evalueret for leveren engraftment, og tilstedeværelsen af humane immunceller i blodet detekteret af flow flowcytometri bekræfter etableringen af det menneskelige immunsystem. Den model, der er udviklet ved hjælp af protokollen beskrevet her ligner flere komponenter af leverskader fra HIV-1-infektion. Dets oprettelse kunne vise sig at være afgørende for undersøgelser af co-infektion med hepatitis virus og for evaluering af antivirale og antiretrovirale lægemidler.
Introduction
Siden fremkomsten af antiretroviral behandling, der har været et betydeligt fald i dødsfald relateret til HIV-1-monoinfektion. Men, leversygdom er opstået som en almindelig årsag til sygelighed hos HIV-inficerede patienter1,2. Coinfektioner af hepatitis virus med HIV-1-infektion er mere almindeligt, tegner sig for 10%-30% af HIV-inficerede personer i USA3,4,5.
Den Host-specificitet af HIV-1 og hepatitis vira begrænser nytten af små dyremodeller til at studere menneskelige-specifikke smitsomme sygdomme eller til at undersøge flere aspekter af HIV-1-associeret lever patogenese. Immundefekt mus, der tillader engraftment af humane celler og/eller væv (betegnet humaniseret musemodeller) er acceptable dyremodeller for prækliniske studier6,7,8. Siden introduktionen af humaniserede mus i begyndelsen af 2000 ‘ erne, flere prækliniske studier af kolestatisk Human levertoksicitet, humane-specifikke patogener, herunder HIV-1 og HIV-associerede neurokognitive lidelser, Epstein Barr virus, hepatitis, og andre smitsomme sygdomme, er blevet undersøgt i disse mus6,9,10,11. Flere musemodeller til CD34+ hspc’er og/eller menneskelig hepatocyt transplantation er længe blevet udviklet og er blevet forbedret med tiden for at studere sygdoms patogenesen af hepatitis B-virus (HBV)-associeret leversygdom12, 13 , 14. flere modeller for HSPC og human hepatocyt (HEP) transplantation er baseret på stammer, kendt som nog (NOD. CG-prkdcscid Il2rgtm1Sug/jictac)8,13, NSG (NOD. CG-Prkdcscid Il2rgtm1Wjl/Szj)15, Balb/C-Rag2-/- γc-/- (Rag2TM 1.1 FLV Il2rgTM 1.1 FLV/j)12, og Fah-/- NOD rag1-/- il2rγnull mus16. Men hver model har sine egne fordele og begrænsninger; for eksempel, AFC8 Dual humaniseret mus til HEPs og humane stamceller (HSCs) på en Balb/C-Rag2-/- γc-/- baggrund muliggør vellykket engraftment af immunceller og hscs, men der er en mangel på en antigen-specifik T-og B-celle respons i denne model12. De største betænkeligheder ved rekonstitution af dobbelte humaniserede mus omfatter suboptimal engraftment, mangel på egnede modeller til at understøtte forskellige væv, uoverensstemmende forhold, immun afvisning eller graft-versus-Host-sygdom (gvhd) og teknisk vanskeligheder, såsom risikable manipulationer med nyfødte og høj dødelighed på grund af metaboliske abnormaliteter13.
Selv om humaniseret mus har været anvendt til hiv-forskning i mange år17,18,19, brugen af humaniserede mus til at studere leverskader forårsaget af HIV-1 har været begrænset20. Vi har tidligere rapporteret etableringen af en dual humaniseret TK-NOG musemodel og dens anvendelse i HIV-associeret leversygdom8. Denne model viser den robuste engraftment af leveren og immunceller og rekapitulerer HIV-smitte patogenese. Denne drøftelse præsenterer en detaljeret protokol, herunder de mest kritiske trin i transplantationen af humane hepatocytter. En beskrivelse af de Hspc’er, der kræves for en vellykket engraftment af HEPs og etableringen af et funktionelt immunsystem i TK-NOG mus er også præsenteret. Brugen af disse mus til at studere HIV-associeret lever immunopogenese er detaljeret. TK-nog hanmus, der bærer en lever specifik herpes simplex virus type 1 thymidinkinase (HSV-tk) transgen anvendes. Mus leverceller, der udtrykker denne transgene kan let være ablateret efter en kort udsættelse for en ikke-toksisk dosis af gcv. Transplanterede humane leverceller holdes stabilt i muse leveren uden udefrakommende lægemidler21. Musene er også konditioneret med nonmyeloablative doser af treosulfan til at skabe en niche i musens knoglemarv for humane celler8. Immunodedygtige TK-NOG-mus injiceres intrasplenisk med HEPs og Multipotente Hspc’er. Musene overvåges derefter regelmæssigt for blod og lever rekonstitution ved blod immunophenotyping og målinger af serum humant-albuminniveauer, hhv. Mus med en vellykket rekonstitution på mere end 15% for både humane immunceller og HEPs er intraperitonealt injiceret med HIV-1. Effekten af HIV på leveren kan vurderes så tidligt som 4-5 ugers post infektion. Det er vigtigt at bemærke, at fordi HIV-1 anvendes, skal alle nødvendige forholdsregler tages, mens håndtering af virus og indsprøjte det i mus.
Protocol
Representative Results
Discussion
Leveren er kompromitteret og beskadiget hos HIV-inficerede patienter24. Eksperimentelle små dyremodeller til at studere menneskelige leversygdomme i nærværelse af HIV-1 er yderst begrænset, på trods af tilgængeligheden af et par cotransplanterede dyremodeller med CD34+ hspc’er og hepatocytter7,12, 25. I in vitro forsøg, hepatocytter er vist at have lav-niveau HIV-1-infektion<sup class="…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af National Institute of Health Grant R24OD018546 (til L.Y.P. og S.G.). Forfatterne vil gerne takke weizhe Li, ph.d., for hjælp i kirurgiske procedurer, Amanda Branch Woods, B.S., Yan Cheng for immunohistologi, unmc flow flowcytometri forsknings facilitets medlemmer direktør Phillip hexley, ph.D., Victoria B. Smith, B.S., og Samantha Wall, b.s., unmc avanceret mikroskopi Core Facility medlemmer Janice A. Taylor, b.s., og James R. talaska, b.s., for teknisk support. Forfatterne anerkender DRs. Mamoru Ito og Hiroshi Suemizu fra CIEA for at give TK-NOG mus og Dr. Joachim Baumgart for at give treosulfan. Forfatterne takker Dr. Adrian Koesters, UNMC, for hendes redaktionelle bidrag til manuskriptet.
Materials
| 27G1/2" needles | BD biosciences | 305109 | |
| 30G1/2" needles | BD biosciences | 305106 | |
| 5 mL polystyrene round-bottom tube 12 x 75 mm style | Corning | 352054 | |
| BD 1 mL Tuberculin Syringe Without Needle | BD biosciences | 309659 | |
| BD FACS array bioanalyzer | BD Biosciences | For purity check of eluted CD34+ cells | |
| BD FACS array software | BD Biosciences | Software to analysis acquired CD34+ cell on FACS array | |
| BD FACS lysing solution | BD Biosciences | 349202 | To lyse red blood cells |
| BD LSR II | BD Biosciences | Instrument for acquisiton of flow cytometry samples | |
| BD Vacutainer Plastic Blood Collection Tube | BD biosciences | BD 367874 | To collect Cord blood |
| Bovine Serum Albumin | Sigma-aldrich | A9576 | |
| Buprenorphine | Controlled substance and pain-killer | ||
| CD14-PE | BD Biosciences | 555398 | Specific to human |
| CD19-BV605 | BD Biosciences | 562653 | Specific to human |
| CD34 MicroBead Kit, human | Miltenyi Biotec | 130-046-702 | For isoation of CD34+ HSPC |
| CD34-PE, human | Miltenyi Biotec | 130-081-002 | Antibody used for purity check of eluted CD34+ cells |
| CD3-AF700 | BD Biosciences | 557943 | Specific to human |
| CD45-PerCPCy5.5 | BD Biosciences | 564105 | Specific to human |
| CD4-APC | BD Biosciences | 555349 | Specific to human |
| CD8-BV421 | BD Biosciences | 562428 | Specific to human |
| Cell counting slides | Bio-rad | 1450015 | |
| ChargeSwitch gDNA Mini Tissue Kit | Thermofisher scientific | CS11204 | for extraction of genomic DNA from ear piece |
| Cobas Amplicor system v1.5 | Roche Molecular Diagnostics | bioanalyzer to measure viral load | |
| Cotton-tipped applicators | McKesson | 24-106-2S | |
| Cytokeratin-18 (CK18) | DAKO | M7010 | Specific to human |
| DMSO (Dimethyl sulfoxide) | Sigma-aldrich | D2650-5X5ML | |
| Extension set Microbore Slide Clamp(s) Fixed Male Luer Lock. L: 60 in L: 152 cm PV: 0.55 mL Fluid Path Sterile | BD biosciences | 30914 | Attached to dispensing pippet and to load with HSPC and HEP suspesion |
| FACS Diva version 6 | BD Biosciences | flow cytometer software required for acqusition of sample | |
| Fetal Bovine Serum (FBS) | Gibco | 10438026 | |
| FLOWJO analysis software v10.2 |
FLOWJO, LLC | flow cytometry analysis software | |
| Ganciclovir | APP Pharmaceuticals, Inc. | 315110 | Prescripition drug |
| Greiner MiniCollect EDTA Tubes | Greiner bio-one | 450475 | |
| Hepatocytes thawing medium | Triangle Research Labs | MCHT50 | |
| Horizon Open Ligating Clip Appliers | Teleflex | 537061 | To hold the ligating clips |
| Hospira Sterile Water for Injection | ACE surgical supply co. Inc. | 001-1187 | For dilution of Buprenorphine (pain-killer) |
| Human Albumin ELISA Quantitation Set | Bethyl laboratories | E80-129 | For assesing human albumin levels in mouse serum |
| Human hepatocyte | Triangle Research Labs | HUCP1 | Cryopreserved human hepatocytes, induction qualified |
| Iris Scissors, Straight | Ted Pella, Inc. | 13295 | |
| Lancet | MEDIpoint | Goldenrod 5 mm | |
| LS columns | Miltenyi Biotec | 130-042-401 | Used to entrap CD34+ microbeads (positive selection) |
| Lymphocyte Separation Medium (LSM) | MP Biomedicals | 50494 | For isoation of lymphocytes from peripheral blood |
| MACS MultiStand | Miltenyi Biotec | 130-042-303 | holds Qudro MACS seperator and LS columns |
| McPherson-Vannas Micro Dissecting Spring Scissors | Roboz Surgical Instrument Co. | RS-5605 | Used to make an incision on skin to expose spleen |
| Micro Dissecting Forceps | Roboz Surgical Instrument Co. | RS-5157 | to hold and pull out spleen from peritoneal cavity |
| mouse CD45-FITC | BD Biosciences | 553080 | mouse-specific |
| PBS (Phosphate Buffered Saline) | Hyclone | SH30256.02 | |
| Qudro MACS separator | Miltenyi Biotec | 130-090-976 | holds four LS columns |
| RPMI 1640 medium | Gibco | 11875093 | |
| StepOne Plus Real Time PCR | Applied Biosystems | Instrument used to genotype | |
| Stepper Series Repetitive Dispensing Pipette 1ml | DYMAX CORP | T15469 | Used to dispense HSPC and HEP supension in controlled manner |
| Suturevet PGA synthetic absorbale suture | Henry Schein Animal Health | 41178 | Suturing of skin and peritoneum |
| TaqMan Gene Expression Master Mix | Thermofisher scientific | 4369016 | |
| TC20 automated cell counter | Bio-rad | 1450102 | |
| TK-NOG mice | Provided by the Central Institute for Experimental Animals (CIEA, Japan; Drs. Mamoru Ito and Hiroshi Suemizu) | ||
| Treosulfan | Medac GmbH | Provided by Dr. Joachim Baumgart (medac GmbH) | |
| Trypan Blue | Bio-rad | 1450022 | |
| Vannas-type Micro Scissors, Straight, 80mm L | Ted Pella, Inc. | 1346 | Used to make an incision on skin to expose spleen |
| Weck hemoclip traditional titanium ligating clips | Esutures | 523700 | To ligate the spleen post-injection |
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