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Biologia do Desenvolvimento

RANKL 小鼠骨髓破骨细胞培养法研究 mTORC1 在破骨细胞形成中的作用

Published: March 15, 2018
doi:
10.3791/56468
, , , , , , ,
1Department of Pediatric Dentistry, Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology,National Clinical Research Center of Stomatology, 2State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology,University of Chinese Academy of Sciences, 3Department of Oral and Maxillofacial-Head and Neck Oncology, Ninth People’s Hospital,Shanghai Jiaotong University School of Medicine

Summary

本手稿描述了一种从小鼠骨髓中分离和培养破骨细胞的体外的协议, 并研究雷帕霉素复合物1在破骨细胞形成中的作用。

Abstract

破骨细胞是独特的骨吸收单元, 区别于单核细胞/巨噬细胞血统的骨髓。破骨细胞的功能障碍可能导致一系列的骨代谢疾病, 包括骨质疏松症。为预防病理性骨质量损失, 制定药物靶点, 必须了解破骨细胞与前体区分的机制。为了确定特定基因在破骨细胞分化中的作用, 在体外分离和培养大量破骨细胞的能力是至关重要的。雷帕霉素复合 1 (TORC1) 在破骨细胞中的哺乳动物/机械靶的失活可减少破骨细胞数量, 增加骨量;然而, 基本机制需要进一步研究。本研究以 RANKL 为基础, 对小鼠骨髓破骨细胞进行分离培养, 研究 mTORC1 灭活对破骨细胞形成的影响。该协议成功地导致了大量的巨型破骨细胞, 通常在一周之内。删除猛禽受损破骨细胞的形成和减少分泌酒石酸耐酸性磷酸酶的活性, 表明 mTORC1 对破骨细胞形成至关重要。

Introduction

骨骼是一个不断变化的器官, 在整个生命中被成骨细胞和破骨鼠重塑。破骨细胞负责矿化基质的吸收和成骨体的合成和分泌新的骨基质1。骨吸收与骨形成之间的平衡是骨骼健康的关键, 包括骨质量的维持和对刺激和损伤的反应。如果这种平衡被打乱, 可能会发生一系列的骨代谢疾病, 包括骨质疏松和牙周疾病。在这些疾病中, 破骨吸收引起的骨量损失超过成骨细胞的骨形成能力2,3。因此, 为了发展治疗骨质疏松等骨骼疾病的药物靶点, 了解破骨细胞的生成和生物学是至关重要的4。

破骨细胞是位于骨表面或附近的独特的巨大多核, 属于单核细胞/巨噬细胞家族的 1.罗格·伊博森 k.j. et 。报告了一种在体外生成破骨样细胞的方法, 其中含有 125-二羟基维生素 D35的培养基。核因子-κ B 配体 (RANKL) 的巨噬细胞集落刺激因子 (M CSF) 和受体活化剂的鉴定是破骨体形成的重要因素, 极大地提高了 osteoclastogenesis 的效率. 1,6,7. 培养破骨细胞体外的能力提高了我们对破骨细胞生成和调控的认识。

雷帕霉素 (mTOR) 的哺乳动物/机械靶在两个结构上和功能上不同的复合体中起作用, 即 mTORC1 和 mTORC28,9。两种多蛋白复合物由于其不同的成分和下游基质而相互区别。mTORC1 包含 mTOR (猛禽) 的独特的调控相关蛋白, 而 mTORC2 包含 mTOR (Rictor)9的雷帕霉素不敏感的伴侣。mTORC1 可以整合和传递重要信号, 调节细胞的生长、增殖和分化。最近, 我们表明, mTORC1 在分解骨吸收网络中发挥关键作用, 通过删除猛禽, 以禁用破骨细胞10中的 mTORC1。然而, 基本机制需要进一步研究。本研究采用 RANKL osteoclastogenic 法从野生型 () 和说唱 Ctsk 小鼠骨髓源性巨噬细胞 (BMMs) 中生成破骨瘤, 并研究 mTORC1 灭活对破骨瘤的影响. 形成.

Protocol

所有与动物有关的程序都是根据斯坦福大学动物护理行政小组 (亚太) 批准的议定书进行的, 并由上海生物化学和细胞研究所动物保育和使用委员会批准。生物学. 1. 准备 生成破骨细胞特定的猛禽删除鼠标 (猛禽fl/佛罗里达州;Ctsk, 此后RapCtsk) 通过交配猛禽fl/佛罗里达州小鼠与Ctsk 的小鼠。在本研究中, 使用<…

Representative Results

使用本议定书, 在6天看到大量的巨型破骨细胞;如果没有发现巨型破骨细胞, 可能需要再多一天的破骨细胞分化 (图 1)。成功的破骨细胞形成通过陷阱染色确认 (图 2A)。破骨细胞是巨大的葡萄酒红/紫色, 有3多个细胞核。在 BMMs (图 4A) 中, 96 井板的每个井中都有超过250只破骨细胞获得。 <p class="jove_content" fo:…

Discussion

osteoclastogenic 检测是最广泛使用的方法, 以分离和培养破骨细胞在体外12,13。虽然已描述了几个基于 RANKL 的破骨细胞归纳13、14、15, 但本研究描述了一种基于先前方法的修改协议。

在上一项研究中, BMMs 在隔离后立即电镀14,<sup class=…

Declarações

The authors have nothing to disclose.

Acknowledgements

作者感谢夏明翰博士和美国加藤的好意提供试剂和老鼠。我们感谢邹实验室的成员进行了有益的讨论。这项工作部分是由中国科学技术部 (大多数) [2014CB964704 和 2015CB964503] 的973项目资助的, 上海交通大学医学院第九人民医院临床研究计划。感谢为细胞生物学核心设施和化学生物学核心设施的帮助, 中国科学院分子细胞科学中心, 上海生物化学与细胞生物学, 中国科学院。

Materials

Raptorfl/fl mice The Jackson Laboratory 013188
Ctsk-cre mice a gift from S. Kato, University of Tokyo, Tokyo, Japan
α-MEM Corning 10-022-CVR
Glutamine Gibico 25030081
Penicillin streptomycin Gibico 15140122
Fetal calf serum BioInd 04-001-1A
Recombinant mouse M-CSF protein R&D Q3U4F9
Recombinant mouse RANKL protein R&D Q3TWY5
RBC lysis buffer Beyotime C3702
Trypan blue Sigma-Aldrich 302643
Acetone Shanghai Chemical Co. Ltd.
Citrate solution Sigma-Aldrich 915
Formaldehyde solution Shanghai Chemical Co. Ltd.
Acid Phosphatase, Leukocyte (TRAP) Kit Sigma-Aldrich 387A-1KT
Fast Garnet GBC Base solution Sigma-Aldrich 3872
Sodium Nitrite Solution Sigma-Aldrich 914
Naphthol AS-BI Phosphate Solution Sigma-Aldrich 3871
Acetate solution Sigma-Aldrich 3863
Tartrate solution Sigma-Aldrich 3873
Dulbecco's phosphate-buffered saline Corning 21-031-CVR
L-tartaric acid Sigma-Aldrich 251380
Sodium tartrate dibasic dehydrate Sigma-Aldrich s4797
Glycine Shanghai Chemical Co. Ltd.
MgCl2 Shanghai Chemical Co. Ltd.
ZnCl2 Shanghai Chemical Co. Ltd.
NaOH Shanghai Chemical Co. Ltd.
Phosphatase substrate Sigma-Aldrich P4744
anti-Raptor Cell Signaling Technology 2280
anti-P-ribosomal protein S6 (S235/236) Cell Signaling Technology 2317
anti-ribosomal protein S6 Cell Signaling Technology 2211
anti-β-actin Santa Cruz Biotechnology sc-130300
37% formaldehyde Xilong scientific
polyvinylidene fluoride (PVDF) membrane Bio-Rad
Western Chemiluminescent HRP Substrate (ECL) Millipore 00000367MSDS
IX71 Olympus
Envision Perkin Elmer
0.45-mm Syringe
Scissor
Mosquito forcep
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Referências

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  2. Jaenisch, R., Bird, A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nature Genetics. 33, 245-254 (2003).
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  4. Boyce, B. F. Advances in osteoclast biology reveal potential new drug targets and new roles for osteoclasts. J Bone Miner Res. 28 (4), 711-722 (2013).
  5. Ibbotson, K. J., Roodman, G. D., McManus, L. M., Mundy, G. R. Identification and characterization of osteoclast-like cells and their progenitors in cultures of feline marrow mononuclear cells. J Cell Biol. 99 (2), 471-480 (1984).
  6. Lacey, D. L., et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell. 93 (2), 165-176 (1998).
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  10. Dai, Q., et al. Inactivation of Regulatory-associated Protein of mTOR (Raptor)/Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling in Osteoclasts Increases Bone Mass by Inhibiting Osteoclast Differentiation in Mice. J Biol Chem. 292 (1), 196-204 (2017).
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  12. Weischenfeldt, J., Porse, B. Bone Marrow-Derived Macrophages (BMM): Isolation and Applications. CSH Protoc. 2008, (2008).
  13. Bradley, E. W., Oursler, M. J. Osteoclast culture and resorption assays. Methods Mol Biol. 455, 19-35 (2008).
  14. Tevlin, R., et al. Osteoclast derivation from mouse bone marrow. J Vis Exp. (93), e52056 (2014).
  15. Xing, L., Boyce, B. F. RANKL-based osteoclastogenic assays from murine bone marrow cells. Methods Mol Biol. 1130, 307-313 (2014).
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  18. Wein, M. N., et al. Control of bone resorption in mice by Schnurri-3. Proc Natl Acad Sci U S A. 109 (21), 8173-8178 (2012).

Tags

Keywords: OsteoclastRANKLMTORC1Bone MarrowIn Vitro CultureOsteoporosisCell IsolationCell Differentiation
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Dai, Q., Han, Y., Xie, F., Ma, X., Xu, Z., Liu, X., Zou, W., Wang, J. A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation. J. Vis. Exp. (133), e56468, doi:10.3791/56468 (2018).
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