Etablering af proliferativ Tetraploide Celler fra transformerede humane fibroblaster
Summary
Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.
Abstract
Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.
Introduction
Polyploidi er observeret ikke kun i specialiserede væv hos pattedyrarter, men også i en række forskellige patologiske tilstande, såsom cancer og degenerative sygdomme. Polyploide (hovedsagelig tetraploide) celler er ofte observeret i præneoplastiske læsioner af humane væv, såsom Barretts øsofagus 1,2 eller skællede intraepithelial læsioner af cervix 3,4, og er blevet anset for at være kilden til maligne aneuploide celler i disse væv 5 6. Selv om det antydes, at omdannelsen af tetraploid til aneuploide celler kunne være en afgørende begivenhed i de tidlige stadier af tumorigenese, er involveret i denne proces mekanismer ikke fuldt forstået. Dette er dels fordi ingen in vitro model har været til rådighed, hvor ikke-transformerede polyploide humane celler kan formere sig.
Nogle forskere har fremkaldt tetraploidy i ikke-transformerede humane epitelceller gennem generering af binucleated celler ved inhibiting cytokinese 7-9. I denne fremgangsmåde er imidlertid unødvendige diploide celler skal elimineres ved fluorescensaktiveret cellesortering (FACS) 7,8 eller kloning ved begrænsende fortynding 9. Fordi disse procedurer er arbejdskrævende og ikke let at udføre, til enklere metoder etablere transformerede tetraploide celler ønskes for forskning på dette område.
I nærværende rapport beskriver vi en protokol til at etablere proliferative tetraploide celler fra normale humane fibroblaster eller telomerase-udødeliggjort humane fibroblaster ved relativt enkle procedurer. Procedurerne bruger spindel gift demecolcin (DC) anholdelse diploide celler i mitose og mitotiske celler indsamlet af ryste yderligere behandlet med DC. Diploide mitotiske celler behandlet med DC i længere tid konvertere til tetraploide G1 celler, og disse celler formere så tetraploide celler efter vækst anholdelse i flere dage efter fjernelse narkotika. Denne protokol giveren effektiv fremgangsmåde til at skabe en nyttig model til at studere forholdet mellem kromosom ustabilitet og den onkogene potentiale polyploide humane celler.
Protocol
Representative Results
Discussion
Et stort problem i induktion af tetraploidy fra diploide celler ved kemiske midler, enten ved cytokinese inhibitorer eller ved tentraadsinhibitor, er, at cellerne bliver ofte en blanding af diploide og tetraploide populationer, og tetraploide celler skal være adskilt fra diploide celler. Mest almindelige tilgange til isolering af en tetraploid befolkning fri for diploide celler bruger FACS eller kloning ved at begrænse fortynding. Men disse procedurer er arbejdskrævende og ikke let at udføre. I denne rapport præsen…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Mrs. Matsumoto for the technical assistance.
Materials
| MEM-α | Sigma-Aldrich | M8042-500ML | |
| Trypsin-EDTA | Sigma-Aldrich | T4174 | |
| FBS | Sigma-Aldrich | 172012-500ML | |
| Demecolcine solution (10 μg/mL in HBSS) | Sigma-Aldrich | D1925-10ML | |
| BD CycleTES Plus DNA Reagent Kits | BD Biosciences | #340242 | For examination of DNA ploidy by flow cytometry |
| Human chromosome multicolor FISH probe 24XCyte | MetaSystems | #D-0125-060-DI | Specialized filter set and software for mFISH analysis are necessary |
| Isis imaging system with mFISH software | MetaSystems | Specialized probe kit is necessary |
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