非形質転換ヒト線維芽細胞から増殖性四倍体細胞の樹立
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
倍数性は、哺乳動物種の特殊な組織でも、癌や変性疾患のような病的状態の様々なだけでなく、観察されました。倍数体（主に四倍体）細胞は、しばしば、頸部3,4のバレット食道1,2または扁平上皮内病変のようなヒト組織の前癌病変において観察され、そしてそれらの組織5における悪性異数性細胞の供給源であると考えられています、6。それは異数体細胞への四倍の変換は腫瘍形成の初期段階において重要な事象である可能性が示唆されていますが、このプロセスに関与するメカニズムは完全には理解されていません。非形質転換倍数体ヒト細胞を伝播することができる場所ないin vitroモデルが利用できなかったので、これは部分的です。
一部の研究者は、INHによって二核細胞の生成を介して非形質転換ヒト上皮細胞に四倍を誘発しました細胞質分裂7-9 ibiting。しかし、この方法では、不要な二倍体細胞は、蛍光活性化細胞選別（FACS）7,8によって除去されなければなりません または希釈9を制限することによってクローニング。これらの手順が面倒で実行することは容易ではないので、非形質転換四倍体細胞を確立するための簡単な方法は、この分野の研究のために望まれています。
本報告書では、比較的簡単な手順で、正常ヒト線維芽細胞またはテロメラーゼ不死化ヒト線維芽細胞から増殖性四倍体細胞を確立するためのプロトコルについて説明します。手順は、有糸分裂で二倍体細胞を逮捕するためにスピンドル毒デメコルチン（DC）を使用し、オフ振とうすることにより収集有糸分裂細胞は、更に、DCで処理されています。長時間DCで処理された二倍体有糸分裂細胞は、四倍体G1細胞に変換し、これらの細胞は、薬物除去後数日間増殖停止した後に四倍体細胞として増殖します。このプロトコルは、提供します染色体不安定性と倍数体ヒト細胞の発癌性との間の関係を研究するための有用なモデルを作成するための効率的な方法。
Protocol
Representative Results
Discussion
化学物質による、細胞質分裂阻害剤によるスピンドル阻害剤のいずれかによって二倍体細胞から四倍の誘導における主要な問題は、細胞がしばしば二倍体と四倍体集団の混合物となり、四倍体細胞は二倍体細胞から分離されなければならないということです。二倍体細胞の自由な四倍体の集団を単離するための最も一般的なアプローチは、希釈を制限することによって、FACSまたはクローニン…
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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