Isolering, kultur og adipogen induktion af stromale vaskulære fraktionsafledte præadipocytter fra museperiatortisk fedtvæv
Summary
Her beskriver vi isolering, kultur og adipogen induktion af stromale vaskulære fraktionsafledte preadipocytter fra museperiaortisk fedtvæv, hvilket muliggør undersøgelse af perivaskulær fedtvævsfunktion og dets forhold til vaskulære celler.
Abstract
Perivaskulært fedtvæv (PVAT) er et fedtvævsdepot, der omgiver blodkar og udviser fænotyperne af hvide, beige og brune adipocytter. Nylige opdagelser har kastet lys over PVAT’s centrale rolle i reguleringen af vaskulær homeostase og deltagelse i patogenesen af hjerte-kar-sygdomme. En omfattende forståelse af PVAT’s egenskaber og regulering er af stor betydning for udviklingen af fremtidige behandlinger. Primære kulturer af periaorta-adipocytter er værdifulde til at studere PVAT-funktion og krydstale mellem periaorta-adipocytter og vaskulære celler. Dette papir præsenterer en økonomisk og gennemførlig protokol til isolering, dyrkning og adipogen induktion af stromale vaskulære fraktionafledte preadipocytter fra museperiaortafedtvæv, hvilket kan være nyttigt til modellering af adipogenese eller lipogenese in vitro. Protokollen skitserer vævsbehandling og celledifferentiering til dyrkning af periaorta-adipocytter fra unge mus. Denne protokol vil udgøre den teknologiske hjørnesten på bænksiden til undersøgelse af PVAT-funktionen.
Introduction
Perivaskulært fedtvæv (PVAT), en perivaskulær struktur sammensat af en blanding af modne adipocytter og en stromal vaskulær fraktion (SVF), menes at interagere med den tilstødende karvæg via dens sekretom parakrinalt1. Som en kritisk regulator af vaskulær homeostase er PVAT-dysfunktion impliceret i patogenesen af hjerte-kar-sygdomme 2,3,4. SVF for adipocytvæv består af flere forventede cellepopulationer, herunder endotelceller, immunceller, mesothelceller, neuronale celler og fedtstamceller og stamceller (ASPC’er)5,6. Det er velkendt, at ASPC’er, der er bosiddende i fedtvævets SVF, kan give anledning til modne adipocytter5. SVF antages at være en kritisk kilde til modne adipocytter i PVAT. Flere undersøgelser har vist, at PVAT-SVF kan differentiere sig til modne adipocytter under specifikke induktionsbetingelser 6,7,8.
I øjeblikket er der to isolationssystemer til isolering af SVF fra fedtvæv, den ene er enzymatisk fordøjelse og den anden er ikke-enzymatisk9. Enzymatiske metoder resulterer typisk i et højere udbytte af nukleerede stamceller10. Til dato er fordelene ved SVF til fremme af vaskulær regenerering og neovaskularisering i sårheling, urogenitale og hjerte-kar-sygdomme blevet bredt demonstreret11, især inden for dermatologi og plastikkirurgi12,13. Imidlertid er de kliniske anvendelsesmuligheder for PVAT-afledt SVF ikke blevet undersøgt godt, hvilket kan tilskrives manglen på en standardiseret metode til isolering af SVF fra PVAT. Formålet med denne protokol er at etablere en standardiseret tilgang til isolering, dyrkning og adipogen induktion af SVF-afledte preadipocytter fra muse-PVAT, der omgiver thorax aorta, hvilket muliggør yderligere undersøgelse af PVAT-funktionen. Denne protokol optimerer vævsbehandling og celledifferentieringsteknikker til dyrkning af periaorta-adipocytter opnået fra unge mus.
Protocol
Representative Results
Discussion
Vi foreslår en praktisk og gennemførlig tilgang til isolering og adipogen induktion af SVF-afledte preadipocytter fra museperiaortafedtvæv. Fordelene ved denne protokol er, at den er enkel og økonomisk. Et tilstrækkeligt antal mus er afgørende for en vellykket isolering, da utilstrækkeligt væv kan resultere i lav SVF-tæthed og dårlig væksttilstand, hvilket i sidste ende påvirker lipogen effektivitet. Derudover er musenes alder en vigtig faktor at overveje, da SVF’s adipogene potentiale falder med alderen. Hur…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev støttet af National Natural Science Foundation of China (82130012 og 81830010) og Nurture-projekterne for grundforskning på Shanghai Chest Hospital (bevillingsnummer: 2022YNJCQ03).
Materials
| 0.2 μm syringe filters | PALL | 4612 | |
| 12-well plate | Labselect | 11210 | |
| 15 mL centrifuge tube | Labserv | 310109003 | |
| 3,3',5-triiodo-L-thyronine (T3) | Sigma-Aldrich | T-2877 | 1 nM |
| 50 mL centrifuge tube | Labselect | CT-002-50A | |
| anti-adiponectin | Abcam | ab22554 | 1:1,000 working concentration |
| anti-COX IV | CST | 4850 | 1:1,000 working concentration |
| anti-FABP4 | CST | 2120 | 1:1,000 working concentration |
| anti-PGC1α | Abcam | ab191838 | 1:1,000 working concentration |
| anti-PPARγ | Invitrogen | MA5-14889 | 1:1,000 working concentration |
| anti-UCP1 | Abcam | ab10983 | 1:1,000 working concentration |
| anti-α-Actinin | CST | 6487 | 1:1,000 working concentration |
| BSA | Beyotime | ST023-200g | 1% |
| C57BL/6 mice aged 4-8 weeks of both sexes | Shanghai Model Organisms Center, Inc. | ||
| Cell Strainer 70 µm, nylon | Falcon | 352350 | |
| Collagen from calf skin | Sigma-Aldrich | C8919 | |
| Collagenase, Type 1 | Worthington | LS004196 | 1 mg/mL |
| Dexamethasone | Sigma-Aldrich | D1756 | 1 μM |
| Dispase II | Sigma-Aldrich | D4693-1G | 4 mg/mL |
| Fetal bovine serum | Gibco | 16000-044 | 10% |
| HEPES | Sigma-Aldrich | H4034-25G | 20 mM |
| High glucose DMEM | Hyclone | SH30022.01 | |
| IBMX | Sigma-Aldrich | I7018 | 0.5 mM |
| Incubator with orbital shaker | Shanghai longyue Instrument Eruipment Co.,Ltd. | LYZ-103B | |
| Insulin (cattle) | Sigma-Aldrich | 11070-73-8 | 1 μM |
| Isoflurane | RWD | R510-22-10 | |
| Krebs-Ringer's Solution | Pricella | PB180347 | protect from light |
| Microsurgical forceps | Beyotime | FS233 | |
| Microsurgical scissor | Beyotime | FS217 | |
| Oil Red O | Sangon Biotech (Shanghai) Co., Ltd | A600395-0050 | |
| PBS (Phosphate-buffered saline) | Sangon Biotech (Shanghai) Co., Ltd | B548117-0500 | |
| Penicillin-Streptomycin | Gibco | 15140122 | |
| Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 115-035-146 | 1:5,000 working concentration |
| Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) | Jackson ImmunoResearch | 111-035-144 | 1:5,000 working concentration |
| Rosiglitazone | Sigma-Aldrich | R2408 | 1 μM |
| Standard forceps | Beyotime | FS225 | |
| Surgical scissor | Beyotime | FS001 |
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