Intrasplenic Transplantation af hepatocytter efter delvis Hepatectomy i NOD. SCID mus
Summary
Vi har beskrevet en protokol for at udføre delvis hepatectomy (PHx) og celle transplantation via milten i Nik. SCID (NIK. CB17-Prkdcscid/J) mus. I denne protokol, er et snit lavet til at afsløre og resect venstre lap af leveren efterfulgt af et andet snit til intrasplenic transplantation af celler.
Abstract
Delvis hepatectomy er en alsidig og reproducerbar metode til at studere lever regenerering og effekten af celle-baserede therapeutics i forskellige patologiske tilstande. Delvis hepatectomy letter også øget engraftment og spredning af transplanterede celler af accelererende neovascularization og celle migration til leveren. Her beskriver vi en enkel protokol for udfører 30% hepatectomy og transplantation af celler i milt fra en ikke-overvægtige diabetikere/svær kombineret immundefekte NOD. SCID (NIK. CB17-Prkdcscid/J) mus.
I denne procedure, er to små snit lavet. Den første indsnit er at eksponere og resect venstre lap af leveren, og en anden lille snit er lavet til at afsløre milt for intrasplenic transplantation af celler. Denne procedure kræver ikke nogen specialiserede kirurgiske færdigheder, og det kan være afsluttet i 5-7 minutter med mindre stress og smerter, hurtigere helbredelse og bedre overlevelse. Vi har demonstreret transplantation af hepatocytter isoleret fra en grøn fluorescerende protein (NGL) udtrykker mus (genetisk modificeret C57BL/6-Tg (UBC-NGL) 30Scha/J), samt hepatocyt som celler af human oprindelse (NeoHep) i delvis hepatectomized hilsen. SCID mus.
Introduction
I øjeblikket er hepatocyt transplantation foreslået som et alternativ til hele organtransplantation for behandling af patienter med alvorlige leversygdomme. Det menes, at det kan slå bro over patienter til hele orgel transplantation1. Ud over allogen hepatocytter2, xenogen hepatocytter3 og hepatocytter stammer fra stamceller4 er også ved at blive undersøgt i dyremodeller. I denne sammenhæng, målsøgende og engraftment er potentialet i de transplanterede celler i modtageren et vigtigt kriterium for celle-baserede terapi i akut leversvigt (AHF).
For at undersøge transplantation af hepatocytter eller hepatocyt-lignende celler5, er AHF lavet i en dyremodel af kirurgisk6 eller farmakologiske7 procedurer, efterfulgt af transplantation af celler. At gøre en AHF dyremodel af farmakologiske reagenser, mange hepatotoxins d-galactosamine8, acetaminophen9, tetrachlormethan10, thioacetamide11, Concanavalin A12, LPS13 , osv., har været brugt. Fra denne liste, hver reagens genererer et unikt sæt af funktioner for AHF, men desværre ingen enkelt reagens efterligner de menneskelige AHF. Derudover AHF induceret af hepatotoxins tager lang tid, der sætter dyr under kronisk stress, og reproducerbare resultater er vanskelig at opnå.
På den anden side den kirurgiske procedure af delvis hepatectomy (PHx) er dygtighed afhængige, og reproducerbare resultater er let at opnå efter udvikler nødvendige færdigheder. For at fremkalde AHF ved kirurgisk indgreb alene, er resektion af mere end 70% af leveren påkrævet; dog transplanterede mindre end en 70% hepatectomy kan stadig udnyttes til at studere engraftment og spredning af celler i leveren til at analysere deres terapeutiske kapacitet under leverskader14. Transplantation af hepatocytter har været udført post hepatectomy gennem bughinden15, hale vene16, leverens vene17eller milt18. I øjeblikket, er leverens vene infusion og intrasplenic transplantation af hepatocytter de foretrukne procedurer, så de er nemmere at reproducere.
I dette papir, har vi beskrevet en procedure for en 30% delvis hepatectomy i NOD. SCID (NIK. CB17-Prkdcscid/J) mus, hvor den venstre lap af leveren er skåret ud. Det er efterfulgt af transplantation af 0,2 millioner normal god landbrugspraksis udtrykker mus (C57BL/6-Tg (UBC-NGL) 30Scha/J) hepatocytter samt menneskelig oprindelse NeoHep19 i milten. Denne fremgangsmåde fører til engraftment af de transplanterede celler i leveren. Denne procedure er den mindst invasive og et minimalt smertefulde teknik.
Protocol
Representative Results
Discussion
Delvis hepatectomy er en etableret teknik for behandlende lever regenerering, og overdreven hepatectomy er rapporteret at efterligne AHF model. Blandt dyremodeller af Husholdningsfolie af aluminium er gnavere, især mus, de mest undersøgte model. For at opnå en leverskade model i mus, op til en 70% hepatectomy er blevet rapporteret med en god overlevelse sats25,26. Men i nøgen og andre immundefekte mus, en 70% hepatectomy blev rapporteret som fatal og dyr død…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af core tilskuddet modtaget fra Institut for bioteknologi, Indiens regering til de nationale Institut for Immunology, New Delhi. Dr. Bhattacharjee nuværende adresse er afdelingen for gastroenterologi, hepatologi og ernæring, children’s Hospital Los Angeles.
Materials
| Gas Anesthesia System | Ugo Basile; Italy | 211000 | |
| Weighing machine | Goldtech ; India | Local Procurement | |
| Biological safety cabinet ( Class I) | Kartos international; India | Local Procurement | |
| Hair Trimmer | Panasonic ; Japan | ER-GY10 | |
| Straight operating scissor with sharp /sharp blades | Major Surgicals; India | Local Procurement | |
| Forceps with Serrations | Major Surgicals; India | Local Procurement | |
| Micro needle holders straight & curved | Mercian ; England | BS-13-8 | |
| 1 ml insulin syringe with 30G *5/16 needles | Dispo Van; India | ||
| 1 ml syringe with 26 G * 1/2 needle | BD ; US | REF 303060 | |
| Nylon Threads | Mighty ; India | (1-0) Local Procurement | |
| MERSUTURES 4-0 Sterilised Surgical Needled Suture | Ethicon, Johnson & Johnson, India | NW 5047 | |
| TRUGUT 76 cm 4-0 absorbable surgical suture | Sutures India Pvt. Ltd; India | SN 5048 | Sterilised Surgical Needled Suture Catgut Chromic |
| Cotton Buds | Pure Swabs Pvt Ltd ; India | Local Procurement | |
| Surgical Tape | 3M India ; India | 1530-1 | Micropore Surgical Tape |
| Microtome | Histo-Line Laboratories, Italy | MRS3500 | |
| Shandon Cryotome E Cryostat | Thermo Electron Corporation ; US | ||
| Confocal laser scanning microscope | Carl Zeiss ; Germany | LSM 510 META | |
| Bright Field Microscope | Olympus, Japan | LX51 | |
| Automated analyser | Tulip, Alto Santracruz, India | Screen Maaster 3000 | Biochemical analyser for liver functional test |
| Flow Cytometer | BD ; US | BD FACSverse | Assesment of presence of cells post transplantation |
| Veet hair removal cream | Reckitt Benckiser , India | ||
| FORANE | Abbott ; US | isoflurane USP 99.9% | |
| Taxim | AlKem ; India | cefotaxime sodium injection | |
| Povidone-Iodine solution | Win-Medicare; India | Betadine | |
| Paraformaldehyde | Himedia; India | GRM 3660 | |
| Iscove's Modified Dulbecco's Medium (IMDM) | Life technologies, Thermo Fisher scientific ; US | 12200-036 | |
| Sucrose | Sigma ; US | S0389 | |
| Tissue-Tek | Sakura; US | 25608-930 | O.C.T compound |
| DAPI | Himedia; India | MB 097 | |
| anti-Albumin goat Polyclonal | Thermo Scientific,Pierce, US | PA126081 | |
| anti-connexin 32/GJB1 Polyclonal | abcam, UK | ab64609-500 | |
| antiGFP rabbit polyclonal | Santa Cruz biotechnology; US | SC 8334 | |
| Alexa Fluor 594 donkey anti-goat | Molecular Probes , Thermo Fisher Scientific ; US | A11058 | |
| Alexa Fluor 488 donkey anti-sheep | Molecular Probes , Thermo Fisher Scientific ; US | A11015 | |
| Alexa Fluor 594 chicken anti rabbit | Molecular Probes , Thermo Fisher Scientific ; US | A21442 | |
| Goat anti rabbit IgG HRP | Invitrogen, Thermo Fisher Scientific; US | 65-6120 | |
| anti-Ki67 antibody | abcam, UK | ab15580 | |
| Antigen Unmasking Solution, Citric acid base | Vector laboratories, US | H-3300 | |
| ProLong Diamond antifade mountant | Life technologies, Thermo Fisher scientific ; US | P36966 | |
| SGOT (ASAT) KIT | Coral Clinical System, India | ||
| SGPT (ALAT) KIT | Coral Clinical System, India | ||
| Alkaline Phosphatase Kit (DEA) | Coral Clinical System, India | ||
| Hematoxylin Solution, Mayer's | Sigma ; US | MHS16 | |
| Eosin Y solution, alcoholic | Sigma ; US | HT110132 | |
| DPX Mountant | Sigma ; US | 6522 | |
| Melonex (Pain Killer) | Intas Pharmaceuticals Ltd; India | Meloxicam injection | |
| DAB enhanced liquid substrate system tetrahydrochloride | Sigma ; US | D3939 |
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