Transforme İnsan Fibroblastlar gelen Proliferatif Tetraploid Hücreleri kurulması
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
Poliploid memeli türlerinin özel dokularda değil, aynı zamanda kanser ve dejeneratif hastalıklar gibi patolojik durumlar, çeşitli sadece gözlenmiştir. Polyploid (çoğunlukla tetraploid) hücreleri genellikle serviks 3,4 Barrett özofagus 1,2 veya skuamöz intraepitelyal lezyonlar gibi insan dokularının preneoplastik lezyonlar gözlenir ve bu dokularda 5 malign anöploidi hücrelerin kaynağı olarak kabul edilmiştir 6. o Anöploid hücrelere tetraploid dönüşüm tümörigenezinin erken evrelerinde çok önemli bir olay olabileceğini öne olmasına rağmen, bu süreçte yer alan mekanizmalar tam olarak anlaşılamamıştır. Bu dönüştürülmemiş polyploid insan hücreleri yaymak nerede hiçbir in vitro model mevcut olmuştur kısmen çünkü.
Bazı araştırmacılar inh ile binükleer hücrelerin üretilmesi dönüştürülmemiş insan epitel hücrelerinde tetraploidi indüklenensitokinezi 7-9 ibiting. Bu yöntemde, bununla birlikte gereksiz diploit hücreleri 7,8 floresans ile aktive edilen hücre tasnif (FACS) ile elimine edilmelidir veya seyreltme 9 sınırlandırılmasıyla klonlama. Bu işlemler gerçekleştirmek için kolay zahmetli ve olmadığından, basit yöntemler dönüştürülmemiş tetraploid hücreler bu alanda araştırma için istenen kurmak.
Bu raporda, biz nispeten basit prosedürlerle normal insan fibroblast veya telomeraz-ölümsüzleştirilmiş insan fibroblastlar gelen proliferatif tetraploid hücreler kurmak için bir protokol açıklar. prosedürler ayrıca DC ile tedavi edilir kapalı sallayarak tarafından toplanan mitoz diploid hücreleri ve mitotik hücrelerin tutuklama mili zehir demekolsin (DC) kullanın. uzun süreli DC ile tedavi diploid mitotik hücreler tetraploid G1 hücrelere dönüştürmek ve bu hücreler ilaç çıkarılmasını takiben birkaç gün süreyle büyüme tutuklanmasının ardından da tetraploid hücreler çoğalırlar. Bu protokol sağlarkromozom istikrarsızlık ve poliploid insan hücrelerinin onkojenik potansiyeli arasındaki ilişkiyi incelemek için yararlı bir model oluşturmak için etkili bir yöntem.
Protocol
Representative Results
Discussion
kimyasal ajanlarla diploid hücrelerinden tetraploidi indüklenmesinde önemli bir sorun, her iki sitokinez önleyicileri ya da aks önleyicileri, hücreler çoğu zaman, diploid ve tetraploid popülasyonlarının karışımı haline ve tetraploit hücreler diploid hücrelerinden ayrılmalıdır. diploid hücrelerin ücretsiz tetraploid nüfusun izolasyonu için en yaygın yaklaşım, sınırlayıcı seyrelti yoluyla FACS ya klonlamadır. Ancak, bu işlemler zahmetli ve gerçekleştirmek kolay değildir. Bu yazıda, nor…
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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