Name: a rankl-based osteoclast culture assay of mouse bone marrow to investigate the role of mtorc1 in osteoclast formation
Uploaded: 2018-03-15T12:30:00.000+00:00
Duration: 9 min 37 s
Description: Watch this Scientific Journal Video about A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation at JoVE.com

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。

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
<ol><li>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, 使用猛禽fl/fl 小鼠作为小波控制。</li>
	<li>有一个带冰块的容器来保持孤立的骨骼。</li>
	<li>
准备培养基。
	<ol>
<li>用1x 谷氨酰胺、青霉素-链霉素和10% 胎小牛血清补充最低必需培养基α (α), 制备α-记忆培养基。</li>
		<li>制备骨髓巨噬细胞诱导介质, 由50毫升的α-记忆介质和脑脊液, 在 20 ng/毫升。</li>
		<li>在50毫升的α记忆培养基中, 分别制备 20 RANKL 的破骨细胞诱导培养基。</li>
	</ol>
</li>
	<li>
在陷阱染色前准备以下缓冲器。
	<ol>
<li>准备解决方案, 包括6.5 毫升丙酮, 2.5 毫升柠檬酸溶液和0.8 毫升 37% (卷/卷) 甲醛溶液。</li>
		<li>准备陷阱染色液。<ol>
<li>Prewarm 去离子水到37°c。</li>
			<li>将100µL 的快速石榴 GBC 碱溶液和100µL 亚硝酸钠溶液加入1.5 毫升微细, 并在三十年代进行温和反转. 让混合物站立2分钟。</li>
			<li>Prewarm 9 毫升去离子水到37°c。添加200µL diazotized 快速石榴石 GBC 基溶液从步骤 1.4.2.2, 100 µL 的萘酚为双磷酸盐溶液, 400 µL 醋酸溶液, 200 µL 的酒石酸溶液。</li>
			<li>在37摄氏度的水浴中保持陷阱染色混合物溶液。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
在培养培养基的疏水阀活动量化前准备以下缓冲区。
	<ol>
<li>准备酒石酸缓冲液 (50 毫升): 2 毫升0.33 米 l-酒石酸, 48 毫升的酒石酸钠二元脱水。将 pH 值调整为4.9。</li>
		<li>制备 4-硝基苯基磷酸二钠 (pNPP) 缓冲液 (200 毫升): 1.502 克甘氨酸, 41 毫克氯化镁2, 27.2 毫克 ZnCl2, 和180毫升去离子水。调整 pH 值为10.4 与 3 M 氢氧化钠和容量到200毫升与去离子水。</li>
		<li>用磷酸酶基质制备 pNPP 缓冲液 (用于一个96井板): 49.37 毫克磷酸酶基底和175µL pNPP 缓冲液1.5.2。</li>
		<li>准备基板缓冲器 (9 mL/96-well 板): 6.24 毫升去离子水, 360 ul 醋酸溶液, 和2.4 毫升酒石酸缓冲液。采用涡流混合, 预热37摄氏度。</li>
		<li>添加 90 ul 的 pNPP 缓冲器与磷酸酶基底 (1.5. 3) 到9ml 预热基板缓冲 (1.5. 4), 使基板混合物和漩涡十年代。</li>
	</ol>
</li>
</ol>2. 解剖 (天-1)
<ol><li>弄死3个月大的女性和说唱Ctsk 小鼠由 CO2分开。通过训练有素的人员执行 CO2吸入适当的设备。</li>
	<li>在烧杯中浸泡6只老鼠, 100 毫升75% 乙醇 (卷/卷) 5 分钟, 以防止细菌污染, 然后放置在解剖板上仰卧位。垂直切开胫骨远端的切口, 用眼科剪刀解剖皮肤。</li>
	<li>用剪刀切开髋关节关节韧带, 将后肢从树干上脱臼。</li>
	<li>切开膝关节关节韧带, 并小心解除膝关节与胫骨和股骨的分离。用剪刀轻轻地取下软组织。</li>
	<li>将一个基因型的所有骨骼放置在一只老鼠的每一个六井板中, 在冰上有2毫升的α记忆。 注意: 为了保持细胞的生存能力, 这些骨骼应该保持在这些条件下少于1小时。</li>
</ol>3. 隔离 (天-1)
<ol><li>用75% 乙醇 (3 毫升) 和其他5口井 (3 毫升) 填充六井板的一个井。</li>
	<li>将一个基因型的所有骨骼在乙醇洗涤十五年代和洗涤骨头5次以α记忆培养基为十年代每次。</li>
	<li>用剪刀切断骨骺, 将0.45 毫米注射器针插入到骨腔中, 用α-记忆介质将骨髓冲洗成50毫升离心管。</li>
	<li>用与骨骼另一端相同的方法冲洗骨腔。重复至少两次, 彻底冲洗骨腔, 直到骨骼苍白。</li>
	<li>离心机骨髓获得细胞颗粒在 800 x g 和4°c 5 分钟。</li>
	<li>将3毫升的红细胞裂解缓冲液放入离心管中, 轻轻并用重悬骨髓。将离心管放在冰上8分钟, 溶解红细胞。</li>
	<li>在离心管中加入6毫升的α-记忆介质, 以阻止细胞裂解。离心机在 500 x g 和4°c 为10分钟和吸入上清。</li>
	<li>并用重悬在3毫升的α-记忆培养基中, 将细胞放入一个六井板中 (通常每井一只老鼠)。</li>
	<li>一夜之间在 5% CO2孵化器孵化37摄氏度。</li>
</ol>4. 电镀 (0 天)
<ol><li>将上清液转移到15毫升离心管, 第二天早上收集不连接的细胞。</li>
	<li>离心机在 800 x g 和4°c 为5分钟和吸入上清。</li>
	<li>并用重悬4毫升的α-记忆培养基。</li>
	<li>取20µL 的细胞溶液, 并与20µL 的台盼蓝混合。将混合物的10µL 添加到计数室, 并获得每毫升溶液的细胞计数。</li>
	<li>添加适当数量的α-记忆培养基, 以获得50万细胞/毫升细胞溶液。</li>
	<li>将500µL 和50µL 骨髓巨噬细胞诱导介质分别添加到24井板和96井板的每个井中。</li>
	<li>分别将500µL 和50µL 的细胞溶液添加到24井板和96井板的每个井中。</li>
	<li>在 5% CO2孵化器中孵育37°c 3 天。</li>
</ol>5. 区别 (天 3-7)
<ol><li>电镀三天后, 从96井板的每个井中收集50µL 介质, 并在-20 摄氏度处冷冻, 然后吸入残余介质。</li>
	<li>分别在24井板和96井板中加入1毫升和100µL 破骨细胞诱导介质入井。</li>
	<li>孵育37°c 2 天。</li>
	<li>从每个井中收集50µL 的培养基, 并吸取残余培养基。</li>
	<li>在每个井中加入破骨细胞诱导介质。</li>
	<li>孵育37°c 1 天。 注意: 在这个时间点, 大的, 多核破骨细胞应该看到在倒置显微镜下 (图2A)。</li>
	<li>如果未发现破骨细胞, 改变破骨细胞诱导培养基, 每天观察直到发现破骨细胞。</li>
	<li>在破骨细胞形成后, 从96井板中的每个井中收集50µL。</li>
</ol>6. 酒石酸耐酸性磷酸酶 (诱捕器) 染色
注: 成熟的破骨细胞在体外培养6-7 天后可能出现 (步骤 4)。
<ol><li>吸入培养基, 并轻轻洗涤3倍 1x PBS。</li>
	<li>用100µL 的固定溶液在室温下修复96井板的每个井中的细胞。</li>
	<li>固定后, 吸入修复液并轻轻地洗涤3次, 用去离子水 prewarmed 到37摄氏度。吸入去离子水。</li>
	<li>在96井板的每个井中添加100µL 的陷印, 并将板材放在孵化器中37°c 30 分钟, 并从光中屏蔽。</li>
	<li>染色后, 吸入陷阱污渍, 并轻轻洗3次与去离子水。</li>
	<li>图像破骨细胞使用倒置显微镜在40X 放大。</li>
</ol>7. 培养基的诱捕活动量化
<ol><li>从步骤5.1、5.4 和 5.8, 从96井板块的每个井中收集30µL 文化上清液;以30µL 非培养型破骨细胞诱导培养基为阴性对照。把样品放在一个新的96井板上。</li>
	<li>添加 90 ul 的基材混合物 (1.5. 5) 入每井96井板材。</li>
	<li>将盘子放在37摄氏度的孵化器中, 1 小时, 然后从光线中屏蔽。</li>
	<li>将50µL 3 米的氢氧化钠加入96井板的每一个井中, 停止反应。</li>
	<li>测量 theabsorbance 在波长405毫微米。</li>
</ol>8. 西方印迹
注: 在 24-井板中6-7 天的体外培养后, 成熟的破骨细胞应可见。
<ol><li>吸入培养基。</li>
	<li>用预冷 1x PBS 轻轻清洗3次。</li>
	<li>吸入 PBS</li>
	<li>添加100µL 含蛋白酶抑制剂鸡尾酒的 1x SDS 裂解缓冲液。</li>
	<li>热裂解物在105°c 10 分钟和离心机在 1.2万 x g 和4°c 为10分钟. 收集含有蛋白质的上清液。</li>
	<li>将含有30µg 蛋白质的裂解物与 10% SDS 页11分开, 然后转移到聚偏氟乙烯 (PVDF) 膜.</li>
	<li>用5% 脱脂牛奶阻挡膜30分钟。</li>
	<li>用抗猛禽, anti-P-S6, anti-S6, 抗β肌动蛋白孵化膜, 在 1:1, 000 稀释5% 脱脂牛奶在4°c 过夜。</li>
	<li>用 Tween-20 (TBST) 在室温下用三缓冲盐水冲洗膜, 10 分钟。</li>
	<li>重复步骤8.9 两次。</li>
	<li>在室温下, 用辣根过氧化物酶 (HRP)-共轭抗鼠或兔 IgG 抗体在 1:5, 000 稀释5% 脱脂牛奶中, 在室温下孵育膜。</li>
	<li>在室温下用 TBST 3 次冲洗膜10分钟。</li>
	<li>在化学发光检测中, 在膜上加入西方化学发光酶基质, 室温孵育5分钟。将多余的衬底排出, 并将污点暴露在 x 射线胶片上。</li>
</ol>

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所有作者都说他们没有利益冲突。

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14,15。我们建议在电镀前一夜培养 BMMs 的基本培养基, 将 BMMs 从骨髓间充质干细胞和成纤维细胞中分离出来, 立即附着在盘子里。脑脊液可以促进单核细胞的增殖, 以破骨的前体和刺激破骨原体表达的等级, 绑定 RANKL, 然后诱导形成成熟的破细胞6,16,17。因此, 在我们的研究中, BMMs 在破骨细胞诱导前3天诱导为 10 ng/毫升脑脊液, 而不是在电镀后第二天为破骨细胞诱导15。细胞密度是关键的破骨干细胞形成在体外。建议将 2.5万 BMMs 在96井板的每一个井中播种, 之后3天的孵化期为 10 ng/毫升脑脊液。当细胞汇合到达80–90% 时, 是 RANKL 的合适时间。通常, 成熟的破骨细胞形成于6天。最常见的原因是少数破骨细胞密度是次优, 这可能是由于低播种密度或低细胞活力的结果。BMMs 是微妙的初级细胞, 需要轻轻的治疗。长期保持骨骼在冰上或 resuspending 有力地降低了 BMMs 的生存能力, 进而损害了破骨细胞的形成。因此, 可行 BMMs 的最佳播种密度对于获得大量破骨细胞是至关重要的。
分泌性诱捕器的活性是破骨吸收的关键。在本研究中, 我们描述了一种定量分析培养基中分泌陷阱活动的方法。培养培养基中诱捕器活性的变化可能是破骨细胞数量或破骨细胞分泌陷阱的改变或两者的结合所致。因此, 这可能是一个有用的方法来分析破骨细胞功能的在体外连同破骨细胞吸收试验描述以前的18。
mTOR 是一种进化保守的蛋白激酶, 可以调节细胞代谢和分化9。通过对破骨细胞形成的调节, 发现 mTORC1 在骨吸收中起重要作用。mTORC1 可能是治疗骨质疏松症等骨代谢紊乱的潜在的药理靶向。
总之, 我们的破骨细胞诱导协议成功地导致了大量的巨型破骨细胞体外, 我们的数据表明 mTORC1 是破骨细胞形成的关键。

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.

<table><tbody><tr><td>&lt;em&gt;Raptor&lt;sup&gt;fl/fl&lt;/sup&gt;&lt;/em&gt; mice</td><td>The Jackson Laboratory</td><td>013188</td><td></td></tr><tr><td>&lt;em&gt;Ctsk-cre&lt;/em&gt; mice</td><td></td><td></td><td>a gift from S. Kato, University of Tokyo, Tokyo, Japan</td></tr><tr><td>&amp;alpha;-MEM</td><td>Corning</td><td>10-022-CVR</td><td></td></tr><tr><td>Glutamine</td><td>Gibico</td><td>25030081</td><td></td></tr><tr><td>Penicillin streptomycin</td><td>Gibico</td><td>15140122</td><td></td></tr><tr><td>Fetal calf serum</td><td>BioInd</td><td>04-001-1A</td><td></td></tr><tr><td>Recombinant mouse M-CSF protein</td><td>R&amp;amp;D</td><td>Q3U4F9</td><td></td></tr><tr><td>Recombinant mouse RANKL protein</td><td>R&amp;amp;D</td><td>Q3TWY5</td><td></td></tr><tr><td>RBC lysis buffer</td><td>Beyotime</td><td>C3702</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma-Aldrich</td><td>302643</td><td></td></tr><tr><td>Acetone</td><td>Shanghai Chemical Co. Ltd.</td><td></td><td></td></tr><tr><td>Citrate solution</td><td>Sigma-Aldrich</td><td>915</td><td></td></tr><tr><td>Formaldehyde solution</td><td>Shanghai Chemical Co. Ltd.</td><td></td><td></td></tr><tr><td>Acid Phosphatase, Leukocyte (TRAP) Kit</td><td>Sigma-Aldrich</td><td>387A-1KT</td><td></td></tr><tr><td>Fast Garnet GBC Base solution</td><td>Sigma-Aldrich</td><td>3872</td><td></td></tr><tr><td>Sodium Nitrite Solution</td><td>Sigma-Aldrich</td><td>914</td><td></td></tr><tr><td>Naphthol AS-BI Phosphate Solution</td><td>Sigma-Aldrich</td><td>3871</td><td></td></tr><tr><td>Acetate solution</td><td>Sigma-Aldrich</td><td>3863</td><td></td></tr><tr><td>Tartrate solution</td><td>Sigma-Aldrich</td><td>3873</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline</td><td>Corning</td><td>21-031-CVR</td><td></td></tr><tr><td>L-tartaric acid</td><td>Sigma-Aldrich</td><td>251380</td><td></td></tr><tr><td>Sodium tartrate dibasic dehydrate</td><td>Sigma-Aldrich</td><td>s4797</td><td></td></tr><tr><td>Glycine</td><td>Shanghai Chemical Co. Ltd.</td><td></td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Shanghai Chemical Co. Ltd.</td><td></td><td></td></tr><tr><td>ZnCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Shanghai Chemical Co. Ltd.</td><td></td><td></td></tr><tr><td>NaOH</td><td>Shanghai Chemical Co. Ltd.</td><td></td><td></td></tr><tr><td>Phosphatase substrate</td><td>Sigma-Aldrich</td><td>P4744</td><td></td></tr><tr><td>anti-Raptor</td><td>Cell Signaling Technology</td><td>2280</td><td></td></tr><tr><td>anti-P-ribosomal protein S6 (S235/236)</td><td>Cell Signaling Technology</td><td>2317</td><td></td></tr><tr><td>anti-ribosomal protein S6</td><td>Cell Signaling Technology</td><td>2211</td><td></td></tr><tr><td>anti-&amp;beta;-actin</td><td>Santa Cruz Biotechnology</td><td>sc-130300</td><td></td></tr><tr><td>37% formaldehyde</td><td>Xilong scientific</td><td></td><td></td></tr><tr><td>polyvinylidene fluoride (PVDF) membrane</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Western Chemiluminescent HRP Substrate (ECL)</td><td>Millipore</td><td>00000367MSDS</td><td></td></tr><tr><td>IX71</td><td>Olympus</td><td></td><td></td></tr><tr><td>Envision</td><td>Perkin Elmer</td><td></td><td></td></tr><tr><td>0.45-mm Syringe</td><td></td><td></td><td></td></tr><tr><td>Scissor</td><td></td><td></td><td></td></tr><tr><td>Mosquito forcep</td><td></td><td></td><td></td></tr></tbody></table>

a rankl-based osteoclast culture assay of mouse bone marrow to investigate the role of mtorc1 in osteoclast formation

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。

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。
对猛禽的删除被西方印迹分析证实, mTORC1 的失活是由核糖体蛋白 S6 磷酸化 (P-S6) 的减少所决定的, 它被广泛用作 mTORC1 的读数 (图 3)。
在RapCtsk BMMs 中, 陷印阳性破骨细胞的数量减少了, 与小波组相比 (图 4A), 这表明猛禽缺陷损害了破骨细胞的形成。
分泌性诱捕器的活性对骨吸收具有重要意义, 对培养培养基中的诱捕活性进行了分析。如图 4B所示, 由于在 RANKL 和RapCtsk BMMs 中激发了对数据的刺激, 陷阱活动增加。此外, RapCtskBMMs 中的陷阱活动减少了, 与相应的重量组 (P < 0.05) 相对应。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56468/56468fig1.jpg"> 图 1: 破骨细胞感应协议的示意图.BMMs 在1天被隔离, 并在一夜之间用基本的α记忆培养基孵化。在0天, BMMs 被收集和镀到一个96井板和24井板后, 在α-记忆介质与 10 ng/毫升脑脊液的孵化。在3天, 培养基被改成α-记忆培养基与 20 ng/ml M 脑脊液和 20 ng/毫升 RANKL 诱导破骨细胞分化。5天, 破骨细胞分化培养基发生了变化。6天, 大量破骨细胞可见, 破骨细胞化验。如果成骨细胞不可见, 则改变了破骨的诱导培养基, 孵化持续了一天。<a href="//cloudflare.jove.com/files/ftp_upload/56468/56468fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56468/56468fig2.jpg"> 图 2: 破骨细胞的代表性视图.(A) 巨型破骨细胞在6天亮场观察到。红线勾勒出破骨细胞的边界。(B) 典型的巨型、多核、陷阱阳性 (红葡萄酒) 破骨细胞。黑色箭头表示多核破骨细胞的两个细胞核。<a href="//cloudflare.jove.com/files/ftp_upload/56468/56468fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56468/56468fig3.jpg"> 图 3: mTORC1 在RapCtsk BMMs 中失活.西方印迹分析猛禽, P-S6 和 S6 的表达在小波和说唱Ctsk BMMs 在6天。<a href="//cloudflare.jove.com/files/ftp_upload/56468/56468fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56468/56468fig4.jpg"> 图 4: 删除猛禽受损破骨细胞的形成.(A) BMMs 6 天的陷阱染色。分别显示了小波和RapCtsk 组中正方形的高放大图像。在RapCtsk BMMs 中, 与组相比, 有较少的巨型、陷阱阳性破骨细胞形成。(B) 培养的小波和RapCtsk BMMs 的陷阱活动. 数据代表的意思是 S.D. * P < 0.05, n = 3。<a href="//cloudflare.jove.com/files/ftp_upload/56468/56468fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation at JoVE.com

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

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。

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

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

a-rankl-based-osteoclast-culture-assay-mouse-bone-marrow-to

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

12.8K 次观看.  National Clinical Research Center of Stomatology. 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。

视频: A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

乳腺癌细胞系外周血单核细胞破的人前临床模型的建立

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

科研

癌症研究

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

在鼠标破骨细胞前体Notch信号的刺激

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

发育生物学

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

一种新型的<em&gt;在体内</em&gt;基因转移技术和<em&gt;在体外</em&gt;细胞分析为肌肉骨骼疾病骨质流失的研究

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

医学

Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro

Bone remodeling is a complex process and it involves periods of deposition and resorption. Bone resorption is a process by which bone is broken down by osteoclasts in response to different stimuli. Osteoclast precursors differentiate into multinuclear osteoclasts in response to macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL). Under pathologic conditions, the cytokine profile is different and involves a mixture of inflammatory cytokines. Tumor necrosis factor alpha (TNF-&#945;) is one of the most important cytokines as it is found in large amounts in areas involved with inflammatory osteolysis. The purpose of this protocol is to provide a method by which murine bone marrow is isolated to generate osteoclasts through induction with M-CSF and either RANKL or TNF-&#945; which will be subsequently inhibited by increasing doses of anti-c-fms antibody, the receptor for M-CSF. This experiment highlights the therapeutic value of anti-c-fms antibody in diseases of inflammatory bone resorption.

This protocol includes dissection of murine long bones and bone marrow isolation, to generate bone marrow macrophages and produce osteoclasts using macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL) or tumor necrosis factor alpha (TNF-&#945;) and their arrest by anti-c-fms antibody, M-CSF receptor.

抗结核抗体对骨细胞形成和骨细胞前体增殖的影响

Bone remodeling is a complex process and it involves periods of deposition and resorption. Bone resorption is a process by which bone is broken down by ...

免疫与感染

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused by increased differentiation and activation of OCs, so the inhibition of this pathobiology is a key therapeutic principle.Two key factors drive the differentiation of OCs from myeloid precursors: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL). Human circulating CD14+ monocytes have long been known to differentiate into OCs in vitro. However, the exposure time and the concentration of RANKL influence the differentiation efficiency. Indeed, protocols for the generation of human OCs in vitro have been described, but they often result in a poor and lengthy differentiation process. Herein, a robust and standardized protocol for generating functionally active mature human OCs in a timely manner is provided. CD14+ monocytes are enriched from human peripheral blood mononuclear cells (PBMCs) and primed with M-CSF to upregulate RANK. Subsequent exposure to RANKL generates OCs in a dose- and time-dependent manner. OCs are identified and quantified by staining with tartrate acid-resistant phosphatase (TRAP) and light microscopy analysis. Immunofluorescence staining of nuclei and F-actin is used to identify functionally active OCs. In addition, OSCAR+CD14&#8722; mature OCs are further enriched via flow cytometry cell sorting, and OC functionality quantified by mineral (or dentine/bone) resorption assays and actin ring formation. Finally, a known OC inhibitor, rotenone, is used on mature OCs, demonstrating that adenosine triphosphate (ATP) production is essential for actin ring integrity and OC function. In conclusion, a robust assay for differentiating high numbers of OCs is established in this work, which in combination with actin ring staining and an ATP assay&#160;provides a useful in vitro model to evaluate OC function and to screen for novel therapeutic compounds that can modulate the differentiation process.

Differentiation of Functional Osteoclasts from Human Peripheral Blood CD14+ Monocytes

Osteoclasts are key bone-resorbing cells in the body. This protocol describes a reliable method for the in vitro differentiation of osteoclasts from human peripheral blood monocytes. This method can be used as an important tool to further understand osteoclast biology in homeostasis and in diseases.

功能性破骨细胞与人外周血CD14+ 单核细胞的鉴别

Osteoclasts (OCs) are bone-resorbing cells that play a pivotal role in skeletal development and adult bone remodeling. Several bone disorders are caused ...

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell numbers. Therefore, the most widely used cellular system is the osteocyte-like MLO-Y4 cell line.
The method here described refers to the use of retinoic acid to generate a homogeneous population of ramified cells with morphological and molecular osteocyte features.
After isolation of osteoblasts from mouse calvaria, all-trans retinoic acid (ATRA) is added to cell medium, and cell monitoring is conducted daily under an inverted microscope. First morphological changes are detectable after 2 days of treatment and differentiation is generally complete in 5 days, with progressive development of dendrites, loss of the ability to produce extracellular matrix, down-regulation of osteoblast markers and up-regulation of osteocyte-specific molecules.
Daily cell monitoring is needed because of the inherent variability of primary cells, and the protocol can be adapted with minimal variation to cells obtained from different mouse strains and applied to transgenic models. 
The method is easy to perform and does not require special instrumentation, it is highly reproducible, and rapidly generates a mature osteocyte population in complete absence of extracellular matrix, allowing the use of these cells for unlimited biological applications.

Treatment of primary mouse osteoblasts with retinoic acid produces a homogeneous population of ramified cells bearing morphological and molecular features of osteocytes. The method overcomes the difficulty of obtaining and maintaining primary osteocytes in culture, and can be advantageous to study cells derived from transgenic models.&#160;

维甲酸的应用获取从初级小鼠成骨细胞骨细胞培养

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

生物学

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

小鼠骨髓破骨细胞的推导

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases (e.g., osteoporosis and bone cancer) and are therefore important objects of research. In vitro, their activity can be analyzed by the formation of resorption pits. In this protocol, we describe a simple pit assay method using calcium phosphate (CaP) coated cell culture plates, which can be easily visualized and quantified. Osteoclast precursors derived from human peripheral blood mononuclear cells (PBMCs) were cultured on the coated plates in the presence of osteoclastogenic stimuli. After 9 days of incubation, osteoclasts were fixed and stained for fluorescence imaging while the CaP coating was counterstained by calcein. To quantify the resorbed area, the CaP coating on plates was stained with 5% AgNO3 and visualized by brightfield imaging. The resorption pit area was quantified using ImageJ.

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Here, we present a simple and effective assay procedure for resorption pit assays using calcium phosphate coated cell culture plates.

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

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

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

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