一种简单的坑式检测方案，用于可视化和定量体外碎屑病再吸收
Summary
在这里，我们提出了一种简单有效的测定程序，用于使用磷酸钙包被细胞培养板进行再吸收坑测定。
Abstract
成熟的破骨细胞是多核细胞，可以通过酸和酶的分泌使骨骼降解。它们在各种疾病（例如骨质疏松症和骨癌）中起着至关重要的作用，因此是重要的研究对象。 在体外，可以通过形成吸收坑来分析它们的活性。在该协议中，我们描述了一种使用磷酸钙（CaP）包被细胞培养板的简单坑测定方法，该方法可以轻松可视化和定量。在存在破骨细胞刺激的情况下，在包被板上培养来自人外周血单核细胞（PBMC）的破骨细胞前体。孵育9天后，固定破骨细胞并进行染色以进行荧光成像，同时将CaP涂层与钙黄绿素复染。为了量化再吸收区域，用5%AgNO3 染色板上的CaP涂层并通过明场成像可视化。使用ImageJ量化了吸收坑区域。
Introduction
破骨细胞（OCs）是来自造血干细胞（HSC）的组织特异性巨噬细胞，与成骨细胞一起在骨重塑中起关键作用1。性激素诱导的、免疫学的和恶性骨骼疾病在全身或局部破坏骨骼是由于骨碎屑活性过大，包括更年期相关的骨质疏松症2、类风湿性关节炎3、牙周病4、骨髓瘤骨病5 和溶骨性骨转移6。相反，OC形成和功能的缺陷也可引起骨石病7。HSC在巨噬细胞集落刺激因子（M-CSF，基因符号ACP5）刺激下分化为OC祖细胞。在M-CSF和NF-κB配体（RANKL，基因符号TNFSF11）的受体激活剂存在的情况下，OC祖细胞进一步分化为单核OC，随后融合成为多核OCs8，9，10。细胞因子M-CSF和RANKL对于诱导破骨细胞标志物是不可或缺的，并且足以诱导骨细胞标志物，例如降钙素受体（CT），核因子κ B（RANK）的受体激活剂，质子泵V-ATP酶，氯化通道7α亚基（CIC-7），整合素β3，酒石酸抗性酸性磷酸酶（TRAP，基因符号ACP5），溶酶体半胱氨酸蛋白酶酶组织蛋白酶K（CTSK）和基质金属蛋白酶9（MMP9）。活化的OC通过形成具有褶皱边界11，12的肌动蛋白环在骨表面上形成密封区。在密封区内，OC通过质子泵V-ATPase 12，13，MMP914和CTSK15分泌质子介导再吸收，导致间隙的形成。
对于体外实验，OC祖细胞可以通过从小鼠股骨和胫骨16，17中扩增骨髓巨噬细胞，以及通过从血液样品和buffy coat18，19，20中分离人外周血单核细胞（PBMC），或通过分化永生的小鼠单核细胞RAW 264.7 21，22来获得。
在本方案中，我们使用衍生自原代PBMCs的OC在CaP包被细胞培养板中描述破破细胞再吸收测定。这里使用的CaP包被细胞培养板的方法采用并从Patntirapong等人17 和Maria等人21先前描述的方法中改进而来。为了获得OC前体，PBMC通过密度梯度离心分离并如上所述20进行扩增。
Protocol
该协议由当地伦理委员会（批准号287/2020B02）审查和批准。 1.磷酸钙包被细胞培养板的制备 钙储备溶液的制备（25 mM CaCl2·2H2O，1.37 mM NaCl，15 mM MgCl2·6H2O在Tris缓冲液中） 准备1.0 M Tris缓冲液，并使用1 M HCl将pH调节至7.4。 在磁力搅拌器上设置玻璃烧杯，加入100 mL 1.0 M Tris缓冲液。 称出0.368克氯化钙2</s…
Representative Results
将细胞培养板底部的磷酸钙包衣分两个包衣步骤进行，包括3天的预钙化和1天的钙化步骤。如图 1所示，在96孔板的底部得到均匀分布的磷酸钙。在执行洗涤步骤后，涂层很好地粘附在底部。 图1：96孔细胞培养板上磷酸钙包衣的代?…
Discussion
在这里，我们描述了一种简单可靠的骨碎屑再吸收测定方法，该方法使用从PBMCs 衍生并在体外 扩增的OC。使用过的CaP包被细胞培养板可以使用实验室可用的材料轻松制备和可视化。除了该方案中采用的未分类的PBMCc外，从小鼠单核细胞21 和骨髓巨噬细胞17 产生的OCs也在类似的合成底物上培养用于坑测定，因此这些细胞源可以移动到参考相应文献的这种方?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作由中国国家留学基金管理委员会（CSC No. 201808440394]）资助。W.C.由CSC资助。
Materials
| AgNO3 | SERVA Electrophoresis GmbH | 35110 | Silver nitrate |
| a-MEM | Gibco | 32561-029 | MEM alpha, GlutaMAX, no nucleosides |
| amphotericin B | Biochrom | 03-028-1B | Amphotericin B Solution |
| CaCl2 | Sigma-Aldrich | 21097-50G | Calcium chloride Dihydrate |
| Calcein | Sigma-Aldrich | C0875 | Calcein |
| FBS | Sigma-Aldrich | F7524 | fetal bovine serum |
| Ficoll | Cytiva | 17144002 | Ficoll Paque Plus |
| Fixation buffer | Biolegend | 420801 | Paraformaldehyde |
| HCl | Merk | 1.09057.1000 | Hydrochloric acid |
| Hoechst 33342 | Promokine | PK-CA707-40046 | Hoechst 33342 |
| M-CSF | PeproTech | 300-25 | Recombinant Human M-CSF |
| MgCl2 | Sigma-Aldrich | 7791-18-6 | Magnesium chloride |
| Na2HPO4 | AppliChem GmbH | A2943,0250 | di- Sodium hydrogen phosphate anhydrous |
| NaCl | Merk | S7653-250G | Sodium chloride |
| NaHCO3 | Merk | K15322429 | Bicarbonate of Soda |
| PBS | Lonza | 17-512F | Dulbecco's Phosphate Buffered Saline (1X), DBPS without Calcium and Magnesium |
| Pen-Strep | Lonza | DE17-602E | Penicillin-Streptomycin Mixture |
| Phalloidin-Alexa Fluor 546 | Invitrogen | A22283 | Alexa Fluor 546 Phalloidin |
| RANKL | PeproTech | 310-01 | Recombinant Human sRANK Ligand (E.coli derived) |
| Tris | Sigma-Aldrich | 93362 | Tris(hydroxymethyl)aminomethan |
| Triton X-100 | Sigma-Aldrich | T8787 | Alkyl Phenyl Polyethylene Glycol |
| TrypLE Express | Gibco | 12605010 | Recombinant cell-dissociation enzymes |
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Cite This Article
Cen, W., Reinert, S., Avci-Adali, M., Alexander, D., Umrath, F. A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro. J. Vis. Exp. (184), e64016, doi:10.3791/64016 (2022).