黑色素瘤细胞衍生因子对骨髓脂肪分化的双重影响
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
在这里, 我们提出了一个可靠和直接的二维 (2D) 共培养系统, 研究肿瘤细胞和骨髓细胞之间的相互作用, 这表明黑色素瘤干细胞衍生因子的双重作用对骨髓脂肪的影响分化, 也为骨转移的机械学研究提供了经典的方法。
Abstract
骨髓脂肪细胞与癌细胞的串扰在骨转移过程中起着至关重要的作用。有多种方法可用于研究显著的串扰;然而, 共培养的二维 transwell 系统仍然是这种串扰研究的经典、可靠、简便的方法。在这里, 我们提出了一个详细的协议, 显示了骨髓脂肪细胞和黑色素瘤的共培养。然而, 这种共培养系统不仅有助于研究骨髓脂肪细胞诱导的癌细胞信号 transductions, 而且可以为今后骨转移的机械学研究提供新的治疗靶点。转移。
Introduction
骨转移在晚期癌症患者中普遍存在, 但治疗仍不可用。除了专门储存能量作为脂肪, 脂肪细胞可以支持肿瘤生长和转移骨髓和其他器官1,2,3,4,5,6。此外, 脂肪细胞在调节癌细胞生物学7、8、9、10和新陈代谢方面起着至关重要的作用,4、11、12 ,13,14,15,16, 以及在骨转移1,4,12。在骨髓小生境中, 脂肪细胞还会影响癌细胞4、6、17的生物学行为。骨髓脂肪细胞与癌细胞 osteotropism 的相互作用对了解骨转移有重要意义。然而, 鲜为人知。
根据目前的研究, 各种方法适用于脂肪细胞, 包括两个或三维 (2/3 d) 和体外培养17,18,19,20,21。最近, Herroon等人设计了一种新的 3 d-培养方法来研究骨髓脂肪细胞与癌细胞的相互作用22。虽然3D 共培养是最佳的模仿脂肪细胞和癌细胞在体内的生理相互作用, 它的重现性差22,23。与2D 共培养系统相比, 3D 共培养系统可以提供不同的细胞表型, 如细胞形态学21、22、24、25、26。此外, 分离的松质骨组织片段的体外培养可以导致骨髓细胞培养出17的生长脂肪体。
然而, 与以前的模型相比, 2D 细胞培养模型仍然是一种经典的、可靠的、易于快速扫描候选分子的技术, 在体外1的脂肪细胞或癌细胞中的表型改变, 4,6,12,15,27.为更好地了解骨髓脂肪细胞与黑色素瘤的相互串扰, 我们提供了一个详细的协议, 为2D 共培养系统的骨髓脂肪细胞与黑色素瘤。
Protocol
注: 本协议中使用的所有细胞在解冻后, 应至少生长三代。 1. 收获黑色素瘤细胞衍生因子 准备 获得 B16F10 细胞和小鼠黑色素瘤细胞系。注: 本协议中, 从中国科学院干细胞库获得了一条小鼠黑色素瘤细胞系。 制作一个完整的培养基为 B16F10 细胞培养 (100 毫升)。使用 Dulbecco 的改良鹰的培养基 (DMEM) 补充10% 胎牛血清 (血清), 50 毫升青霉素和50…
Representative Results
在骨髓, 脂肪细胞可以出现在肿瘤微环境1,13,33,34,35在早期阶段支持肿瘤进展通过可溶性因素或激活 osteoclastogenesis6,12,36, 特别是在肥胖6,12</s…
Discussion
Cocultures 与插入物已被广泛用于研究细胞间的相互作用。2D 共培养系统是观察两个部位在体外进行串扰的有效方法, 我们在这里显示了两种不同的癌细胞驱动对骨髓脂肪细胞的影响。许多实验室利用这种方法来研究脂肪细胞与癌细胞6、12、27、39之间的串扰。
因此, 一般而言,…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢 Dov Zipori (魏茨曼科学研究所, 雷霍沃特, 以色列) 亲切地为我们提供小鼠骨髓基质细胞线14F1.1。这项研究得到了中国国家自然科学基金 (81771729 号) 和重庆医科大学永川医院的资助。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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Citazione di questo articolo
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