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Ricerca sul cancro

デュアル骨髄脂肪細胞分化に及ぼす悪性黒色腫細胞由来因子

Published: August 23, 2018
doi:
10.3791/57329
, , ,
1Department of Rheumatology, Ren Ji Hospital, School of Medicine,Shanghai Jiao Tong University, 2Department of Nephrology and Rheumatology,Yongchuan Hospital of Chongqing Medical University, 3Department of Medical Oncology, Fudan University Shanghai Cancer Center; Department of Oncology, Shanghai Medical College,Fudan University

Summary

ここでは、提案する、信頼性が高く、簡単な二次元 (2 D) 共培養系における骨髄脂肪細胞の悪性黒色腫細胞由来因子の二重効果を明らかにする腫瘍細胞と骨髄脂肪細胞間の相互作用の分化と骨転移機構の解明のための古典的な方法をまたポーズします。

Abstract

骨髄脂肪細胞と腫瘍細胞間クロストークは、骨転移の過程において重要な役割を再生可能性があります。さまざまな方法があります; 重要なクロストークを勉強ただし、共の二次元 transwell システムは古典的な信頼性の高い、このクロストークの研究のための簡単な方法。ここでは、骨髄の脂肪細胞と悪性黒色腫細胞の培養を示す詳細なプロトコルを提案する.それにもかかわらず、このような培養システム可能性がありますだけでなく骨髄脂肪細胞によるがん細胞の細胞シグナル伝達の研究に貢献、骨の新しい治療上のターゲットを明らかにすることが骨転移の研究機構の将来にも転移。

Introduction

骨転移は進行癌患者の間で広まっているが、根治療法はまだ使用できません。脂肪としてエネルギーを格納することに特化したを超えて脂肪細胞は骨髄や他臓器1,2,3,4,5,6で腫瘍の成長と転移をサポートできます。また、脂肪細胞はがん細胞生物学7,8,9,10と代謝4,11,12 の調節に重要な役割を果たしてください。 ,13,14,15,16、よくように骨転移1,4,12として。骨髄ニッチで脂肪細胞は癌細胞4,6,17の生物学的態度も影響します。骨髄脂肪細胞と癌 osteotropism 細胞間の相互作用は骨転移の理解のために重要です。しかし、少しは知られています。

現在の研究に基づいて、さまざまな方法は、2 または 3 次元 (2 D)/前のヴィヴォ文化17,18,19,20,21を含む脂肪細胞に適用されます。最近、Herroonらは、癌細胞22骨髄脂肪細胞の相互作用を研究する新しい 3 D 文化のアプローチを設計されています。3 D 共が生理学的な模倣するため最適な脂肪細胞と癌との間の相互作用が生体内で細胞、それは再現性に乏しく22,23から苦しんでいます。2次元培養システムと比較して、3 D の共培養系細胞形態21,22,24,25,26などの別の細胞表現型があります。さらに、孤立した海綿骨の組織片の前のヴィヴォ文化は、培養した骨髄細胞17から脂肪細胞の堅牢な副産物につながります。

これらの以前のモデルとは対照的しかし、2次元細胞培養モデル古典的な信頼性、および簡単な手法のままさっと候補分子と表現型の脂肪細胞やがんの変更細胞の in vitro1、 4,6,12,15,27.骨髄脂肪細胞と悪性黒色腫細胞間クロストークを理解、悪性黒色腫細胞の骨髄脂肪細胞の 2次元培養システムの詳細なプロトコルを提供します。

Protocol

注: このプロトコルで使用されているすべてのセルは冷凍ストック細胞から解凍後少なくとも 3 世代にわたる栽培にする必要があります。 1. 悪性黒色腫細胞由来因子を収穫します。 準備 B16F10 細胞およびマウスのメラノーマ細胞株を取得します。注: このプロトコル マウス黒色腫細胞株は中国科学院の幹細胞バンクから得られました。 …

Representative Results

骨髄、脂肪細胞が腫瘍微小環境1,13,33,34,35に可溶性因子を介して腫瘍の進行をサポートするための初期段階で表示できるか破骨細胞形成6,12,36, 特に肥満6,</…

Discussion

挿入して共培養系は、細胞間相互作用の研究に広く使用されています。2次元培養システムは、どのように二つの我々 はここで骨髄脂肪細胞の 2 つの異なった癌セル駆動の効果を示した生体外クロストーク、部分を観察する効果的な方法です。多くのラボでは、脂肪細胞と癌細胞6,12,27,39間?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

我々 は骨髄間質細胞をご提供するため親切ドヴ ・ Zipori (ワイツマン科学研究所、レホヴォト、イスラエル) に感謝 14F1.1 ラインします。本研究は、中国国家自然科学基金 (第 81771729)、永川病院の重慶医科大学 (Nos からの補助金によって支えられました。YJQN201330;YJZQN201527)。

Materials

DMEM Invitrogen Inc. 11965092
Fetal Bovine Serum Invitrogen Inc. 16000–044
Phosphate Buffered Saline Invitrogen Inc. 14190-144
Insulin Sigma-Aldrich 91077C
3-isobutyl-1-methyl-xanthine Sigma-Aldrich I5879
Dexamethasone Sigma-Aldrich D4902
Oil Red o Sigma-Aldrich O0625
24-well plate Corning CLS3527
Transwell insert Millipore MCHT24H48
Penicillin/Streptomycin Invitrogen 15140-122
isopropanol Sigma-Aldrich I9516
0.25% trypsin Thermo Scientific 25200056
hemocytometer Bio-Rad 1450016
Culture incubator Thermo Scientific
50ml falcon Corning CLS430828
Clean Bench Thermo Scientific –
Microscopy Olympus –
200 μL pipet tips BeyoGold FTIP620
1000 mL pipet tips BeyoGold FTIP628
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Tags

MelanomaBone MarrowAdipocyte DifferentiationBone MetastasisCell CultureConditioned MediumStromal CellsAdipogenic InductionAdipogenic Maintenance
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Wang, J., Wen, J., Chen, X., Chen, G. Dual Effects of Melanoma Cell-derived Factors on Bone Marrow Adipocytes Differentiation. J. Vis. Exp. (138), e57329, doi:10.3791/57329 (2018).
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