作者想感谢尚塔尔 cro 的技术援助。 这项研究的经费来自 "科特布斯 Atomique 和辅助能源替代品" (URANOs-方案横向 de Toxicologie 杜-cea 和 CPRR cea-阿海珐), 以及来自国际抵抗力量 (铀的毒性: 矿的多级方法过程中的骨骼, ANR-16-CE34-0003)。这项工作也得到了尼斯大教堂-安蒂波利斯和 CNRS 大学的支持。

1. 醋酸铀溶液的制备
<ol><li>要准备2毫升的一个100毫米铀醋酸溶液, 添加85毫克的铀乙酸酯 (上2(OCOCH3)2, 2H2O;M = 424 克. 摩尔-1) 固态到5毫升塑料管。</li>
	<li>将2毫升蒸馏水加入塑料管, 并将其与塑料塞子配合。</li>
	<li>用力摇动管子, 直到固体完全溶解为止。把溶液放到冰箱里24小时。 警告: 铀 (VI) 的操作会带来一些化学和放射性的危害。所有的样品都要准备好, 并在特定的实验室中具有特殊的设备。所有含有 U (VI) 的液体应在专门分配的废物容器中收集, 并作为有毒废料处理。干废料 (吸管、管、等) 可以用 0.1 N 硝酸3解决方案进行清洗, 这将溶解和删除 U (VI)。</li>
	<li>使用微电极和 ph 计验证溶液的 ph 值。 注: 微电极应事先使用商用缓冲液 (ph = 4 和 ph = 7) 进行校准。</li>
</ol>2. 原生264.7 细胞培养
<ol><li>
细胞的维护。
	<ol>
<li>培养原始的264.7 细胞 (ATCC, TIB-71) 在 75 cm2烧瓶在以下成长媒介: Dulbecco 的修改过的老鹰的培养基 (DMEM) 补充5% 胎牛血清和抗生素: 100/毫升青霉素, 100 µg/毫升链霉素。保持细胞在37° c, 5% CO2的孵化器。</li>
		<li>每2到3天更换培养基。当细胞是85-90% 汇合, 机械地从培养瓶去除他们用细胞刮板 (建议由 ATCC) 和分裂他们以比率1:6。 注: 只有3至10通道之间的细胞被用于分化实验。</li>
	</ol>
</li>
	<li>
实验用细胞的制备
	<ol>
<li>如上文所述, 从烧瓶基质中取出细胞, 并将其转移至无菌50毫升管。混合细胞的移向上和向下, 以打破团簇。用例。预计每 75 cm2烧瓶中有35-40 百万单元格。</li>
		<li>计算每个实验所需的细胞悬浮量如下: 实验条件数 x 3 (triplicates) x 1 毫升/井 + 3-4 毫升 (移损失)。 注: 本协议中描述的所有化验的播种密度为5000单元/cm2。因此, 对于一个24孔板, 它意味着1万细胞每井1毫升的培养基, 和细胞悬浮密度因此1万细胞/毫升。</li>
		<li>准备必要的容积1万细胞/mL 细胞悬浮和种子的细胞成 24-井板。对于细胞毒性测定, 使用正常生长培养基。对于鉴别和吸收测定, 使用α修饰的最低必需培养基 (αMEM) 补充2毫米 l-谷氨酰胺, 5% FBS 和 50 ng/毫升的 GST-RANKL17。 注意: 在内部生产的 GST-RANKL 蛋白可以被任何商业上可用的 RANKL 细胞因子所取代。当使用一个新的 RANKL 批次, 它可能是有用的测试它的有效性诱导破通过执行剂量反应实验, RANKL 浓度范围从20到 150 ng/毫升。</li>
	</ol>
</li>
</ol>3. 在培养基中稀释100毫米铀乙酸酯溶液
注: 铀离子 [U (VI)] 在培养基中形成多个与其它介质成分的复合物, 可改变其细胞毒性18,19,20,21。因此, 铀的介质应该即兴, 而不需要省略或缩短平衡步骤。
<ol><li>选择铀暴露浓度并计算实验所需的培养基量 (考虑到每个条件都是三份)。</li>
	<li>从不育细胞培养试验 7.5% 碳酸氢钠水溶液 ([小贩3-] = 893 mm) 开始, 在水中准备一个 100 mm 小贩3解决方案, 并在冰上冷却。</li>
	<li>添加1体积的铀乙酸 100 mm 库存解决方案, 以9卷冰冷 100 mm 小贩3-解决方案, 轻轻摇晃搅拌管。这项操作将稀释铀乙酸酯库存溶液 ([22 +] = 100 mm, ph 4) 到 10 mm, 并带来 pH 值约7。</li>
	<li>添加1卷无菌水到9卷冰冷100毫米小贩3-解决方案达到 90 mm 小贩3(等于小贩 3-集中在 10 mm 铀溶液中), 这将有助于准备控制中.</li>
	<li>允许准备好的解决方案在室温 (RT) 中站立3小时以平衡。</li>
	<li>同时, 准备基本曝光培养基: αMEM 2 毫米 l-谷氨酰胺和 5% FBS。 注: 为鉴别和吸收分析, 添加 50 ng/毫升的 GST-RANKL 的基本曝光介质。</li>
	<li>3小时后, 稀释适量的10毫米铀溶液滴在曝光介质中, 以达到所需的铀工作浓度。通过在最浓缩的铀处理条件下, 在接触介质中加入碳酸氢盐的用量, 同时制备控制介质。</li>
	<li>允许准备好的介质在 RT 处进行3小时的平衡。</li>
	<li>从油井中取出生长介质, 并将其替换为控制和含铀的介质。</li>
</ol>4. 在 U (六) (MTT 细胞毒性测定) 存在的情况下对原始264.7 前体的活性分析
注意: MTT 和亚甲基亚砜均可引起皮肤和眼部刺激。在处理时戴上手套和眼睛保护。在专门分配的废物容器中收集废品。
<ol><li>在 PBS 中溶解适量的 3-[45-dimethylthiazol-2-基]-25-diphenyltetrazolium 溴化铵 (MTT) 粉末, 以获得5毫克/毫升的10x 库存溶液。通过过滤与0.22 µm 过滤器和商店等分在-20 ° c 消毒它。</li>
	<li>24孔板上的平板单元 (每个实验条件下每片3口)。不要忘记为分光光度计坯料预留3空井。</li>
	<li>在铀暴露之前, 在37° c 的22-24 小时孵育细胞, 让细胞附着。</li>
	<li>如3节所述, 准备含铀的介质。</li>
	<li>用含有铀的培养基取代细胞种子板中的生长培养基。孵育24小时。</li>
	<li>计算 1x MTT 溶液的必要容积如下: (实验条件 + 空白) x 3 (triplicates) x 0.4 毫升/井 + 10% 卷移损耗。</li>
	<li>在不含酚红的鹰的最小必需介质 (EMEM) 中稀释10x 的 mtt 溶液, 制备所需的 1x mtt 溶液体积。 注意: 对于成功的色度测定, mtt 应保护不受光照。为了限制背景信号, 建议将 MTT 稀释为 EMEM, 而不是 DMEM。</li>
	<li>从水井中取出含有铀的介质, 用 PBS 清洗, 并在每口井中添加400µL 的 MTT 溶液 (包括3空井)。在黑暗中孵育2.5 小时在37° c。在显微镜下验证暗紫色甲晶体在细胞内形成。</li>
	<li>丢弃 MTT 溶液, 并添加220µL 的二甲基亚砜 (亚砜) 每井。用铝箔包板来保护它们免受光照, 轻轻搅动10分钟以溶解甲晶体。</li>
	<li>使用微光谱仪读取器确定光学密度 (OD) 在 570 nm (甲特异) 和 690 nm (背景)。对于每个井, 减去 690 nm od 570 nm od, 以适应可能的非特定 od 值波动, 由于划痕或混浊22。</li>
	<li>通过从上一步获得的 od 中减去平均外径, 计算出各井的毛坯校正 od。确定每个实验条件的平均 OD 值。</li>
	<li>设置控制条件的平均 OD ((将22 +) = 0 mM) 到100% 的可行性, 并计算其他经测试的铀浓度的相应百分比的细胞活力。绘制剂量响应曲线。评估细胞毒性指数 50 (CI50), 定义为在24h 暴露后导致50% 细胞死亡的铀浓度。</li>
</ol>5. U (VI.) 存在破骨细胞分化的分析
<ol><li>
在铀和疏水阀 (抗酒石酸酸性磷酸酶) 染色的情况下, 对原264.7 细胞进行分化。
	<ol>
<li>在24孔板上板生264.7 细胞, 每实验条件3口。在37° c 孵育约6小时, 准备和平衡含铀的曝光介质的时间。</li>
		<li>如3节所述, 准备具有所需铀浓度的分化介质 (不要忘记向介质添加 GST-RANKL)。</li>
		<li>用含铀的培养基轻轻地替换井中的基本培养基。这一天被认为是0天的破分化。</li>
		<li>通过重复步骤5.1.2 和 5.1.3, 改变2或3天的破分化培养基。核破骨细胞应出现在3和4天之间。</li>
		<li>按照制造商的说明, 用市面上可用的白细胞酸性磷酸酶试剂盒进行诱捕染色。 注: 酒石酸耐酸性磷酸酶 (TRAP) 也称为酸性磷酸酶 5, 抗酒石酸 (ACP5) 在成熟的破骨细胞中被高度表达, 广泛用作破骨细胞分化的标志物。因此, 进行陷阱染色, 以可视化破骨细胞。根据实验条件和破率, 可以在4天或5天的分化过程中进行。</li>
		<li>在4° c 的 PBS 中保持陷阱染色的水井作进一步分析 (它们可以在这些条件下维持3或4周)。</li>
	</ol>
</li>
	<li>
破骨细胞大小/形态分析。
	<ol>
<li>获取每个井的整个表面的图像。图像的分辨率应该足以区分细胞核。考虑具有3或更多核的陷阱阳性细胞, 作为破骨。</li>
		<li>在 ImageJ 软件中打开一个井的图像: "文件" → "打开"。</li>
		<li>验证图像左上角的刻度单位。对于相对量化, 可以使用任何单位。如果破骨细胞大小的绝对值是必要的, 将刻度单位转换为µm: "图像" → "属性"。检查弹出窗口中的 "全局" 框, 以便将新的刻度单位应用于以后打开的所有图像。 注意: 如果使用显微镜进行图像采集, 则在成像软件的每个目标中都显示µm 的像素大小。</li>
		<li>指定要分析的测量参数: "分析" → "设置测量"。在 "设置测量" 窗口中, 检查 "区域" 和 "显示标签" 框, 并取消选中所有其他框。</li>
		<li>打开 roi (感兴趣区域) 经理: "分析" → "工具" → "roi 管理器"。</li>
		<li>在主窗口中, 选取矩形选择工具。在图像的任何地方使用它勾勒出大约 1000 x 1000 µm 的区段。这个选择现在定义了破骨细胞的大小将被分析的第一个区域。</li>
		<li>在 roi 管理器窗口中, 单击 "添加" 按钮, 将感兴趣的区域添加到 roi 管理器中, 然后单击 "更多" → "保存" 以保存它。现在, 通过点击 "更多" → "打开", 可以随时将已保存的 roi 重新加载到 roi 管理器。</li>
		<li>重复步骤 5.2.5-5.2.6 为破骨细胞大小分析定义4附加区域 (图 2A和 2D)。 注: 所定义的区域将用于分析所有图像中破骨细胞的大小。</li>
		<li>创建要分析的5区域之一的副本: 在主要的 roi 管理器中选择此 roi (相应的选择将出现在主映像上) →右键单击选择→ "复制"。使用此副本进行进一步分析。</li>
		<li>在 ROI 管理器窗口中, 复选框 "显示 Al" 和 "标签"。</li>
		<li>在主窗口中, 选取 "多边形选择" 工具, 在选区中手动勾勒一个破骨细胞。通过在 roi 管理器窗口中单击 "添加", 将此大纲添加到 roi 管理器中。对完全位于所选区域 (图 2B和 2E) 内的所有破骨细胞进行类似的操作。</li>
		<li>在 ROI 管理器窗口中, 选择所有的轮廓并通过单击 "测量" 来测量它们的曲面。将在新窗口 (结果窗口、图 2C和2F) 中出现的度量转移到电子表格中。 注: 此时, 与所有破骨细胞轮廓分析的区域可以保存为独立图像: "文件" → "另存为" 的选择格式。</li>
		<li>对当前图像分析的其余区域重复步骤 5.2.8-5.2.12。然后, 对所有其他图像重复整个过程。</li>
	</ol>
</li>
	<li>
破骨细胞数分析与 ImageJ 软件。 注: 由于破骨细胞在井中的分布很少是均匀的, 所以在整个井上进行计数, 而不是在少数选定的区域。由于破骨细胞在形态和大小上都是异构的, 因此没有自动计数单元法是可行的。<ol>
<li>在 ImageJ 软件中打开一个完整的图像 (用于此分析, 使用以前的子节中的图像)。</li>
		<li>打开单元格计数器窗口: "插件" → "分析" → "细胞计数器"。单击 "初始化" 并检查 "类型 1" 框 (或您喜欢的任何类型的数字)。验证 "显示号码" 框是否已选中, 并且 "删除模式" 框未选中。</li>
		<li>在图像上, 点击一个破骨细胞: 有色点和数字将出现在点击。进行类似的所有破骨细胞。通过单击 "单元格计数器" 窗口中的 "保存标记" 来定期保存标记 注意: 如果必须从计数中删除最后一个点, 请单击 "单元格计数器" 窗口中的 "删除"。如果要移除多个点, 请选中 "删除模式" 框, 并通过单击来指示所有无效点。然后取消选中 "删除模式" 框以继续计数。</li>
		<li>通过单击 "单元格计数器" 窗口中的结果来显示计数结果。将最终计数结果转移到电子表格。</li>
	</ol>
</li>
</ol>6. U (VI.) 存在下破骨细胞功能的分析
<ol><li>
坑吸收法。
	<ol>
<li>在24井骨检测板上板生264.7 细胞, 它提供了一种可以吸收破骨的合成无机 osteomimetic 表面。 注意: 为了让细胞附着在仿生表面上, 在添加分化培养基之前, 通常最好把它们放在前一天。</li>
		<li>按照5节 (步骤 5.1.2-5.1.4) 的描述将原始的246.7 细胞分化成破骨。</li>
		<li>在破5天, 通过用10% 漂白溶液取代含铀的培养基, 去除骨模拟表面的破骨细胞。室温孵育5分钟。用去离子水冲洗两次井, 让它们风干。</li>
		<li>将1% 茜素红 S 钠盐溶液添加到油井中, 在非吸收地区染色钙盐。10-15 秒后取出茜素溶液, 用清水轻轻冲洗3-4 次井。让水井风干。 注: 上述染色方法旨在增强吸收与非吸收区的对比度, 以利于连续分析。这一过程必须小心, 因为如果水井没有完全干燥前染色, 其余的骨模拟表面的部分可能会被意外删除。此外, 如果茜素溶液在井中留得太长, 则会出现持久性的非特异染色。应当指出, 本节所述的试验涉及铀对破分化和功能的影响。为了研究铀对破骨细胞形成的影响, 可以用含铀的培养基进行24小时的治疗, 只有一旦成熟的破骨形成 (在分化过程的4或5天) 23。</li>
	</ol>
</li>
	<li>
坑吸收分析。
	<ol>
<li>获取骨检测板各井的整体表面的图像。 注意: 图像采集方法可能不同, 但在整个复制实验中应该是一致的。它们可以包括一幅用固定相机的宏观目标拍摄的图片、高分辨率的扫描图或用自动高吞吐量显微镜制作的马赛克图像。</li>
		<li>从上一节中重复步骤5.2.2 到5.2.4 开始分析。</li>
		<li>将 RGB 图像转换为灰度8位格式: "图像" → "类型" → "8 位"。</li>
		<li>在主窗口中, 选择 "椭圆形选择" 工具。用它画一个圆圈, 勾勒出井的所有表面, 以便进行吸收。</li>
		<li>在步骤5.2.6 中保存新的 ROI。</li>
		<li>为方便阈值的选择, 清除所定义 ROI 之外的所有特征: "编辑" → "清除外部"。 注意: 在这一点上, 通常最好制作一些图片的副本: 取消选择投资回报率, 点击外部的区域→右键点击图片→ "复制"。如果下一步中选择的阈值不理想, 此操作将有助于避免重复上述图像处理。</li>
		<li>选择 "图像" → "调整" → "阈值"。在 "阈值" 窗口中, 使用滑动条选择合适的阈值。所有吸收区域应低于阈值 (白色) 和非吸收, 高于 (红色)。要完成阈值选择, 请单击 "阈值" 窗口中的 "应用"。</li>
		<li>保存最终的二进制图像: "文件" → "另存为" →您选择的格式。</li>
		<li>定义要在感兴趣区域中采取的测量: "分析" → "设置测量"。在 "设置测量" 窗口中, 检查 "区域"、"区域分数" 和 "限制到阈值" 框, 并取消选中所有其他项。</li>
		<li>为了得到 "吸收" 区域的分数直接在结果窗口, 倒置二进制图像 ("编辑" → "倒置")。确保在最终的二进制图像上选择主要的 ROI (否则吸收的区域分数将根据整个图像窗口的表面进行计算)。通过在 ROI 管理器窗口中单击 "度量" 或 "分析" → "度量" 来测量吸收区域分数。保存结果。</li>
	</ol>
</li>
</ol>

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+osteoclast+and+its+unique+cytoskeleton.">The osteoclast and its unique cytoskeleton.</a> Ann N Y Acad Sci. 1240, 14-17 (2011).</li><li>Nesbitt, S. A., Horton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trafficking+of+matrix+collagens+through+bone-resorbing+osteoclasts.">Trafficking of matrix collagens through bone-resorbing osteoclasts.</a> Science. 276 (5310), 266-269 (1997).</li><li>Salo, J., Lehenkari, P., Mulari, M., Metsikko, K., Vaananen, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+osteoclast+bone+resorption+products+by+transcytosis.">Removal of osteoclast bone resorption products by transcytosis.</a> Science. 276 (5310), 270-273 (1997).</li><li>Pierrefite-Carle, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+natural+uranium+on+the+UMR-106+osteoblastic+cell+line:+impairment+of+the+autophagic+process+as+an+underlying+mechanism+of+uranium+toxicity.">Effect of natural uranium on the UMR-106 osteoblastic cell line: impairment of the autophagic process as an underlying mechanism of uranium toxicity.</a> Arch. Toxicol. 91 (4), 1903-1914 (2017).</li><li>Ubios, A. M., Guglielmotti, M. B., Steimetz, T., Cabrini, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uranium+inhibits+bone+formation+in+physiologic+alveolar+bone+modeling+and+remodeling.">Uranium inhibits bone formation in physiologic alveolar bone modeling and remodeling.</a> Environ. Res. 54 (1), 17-23 (1991).</li><li>Bozal, C. B., Martinez, A. B., Cabrini, R. L., Ubios, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+ethane-1-+hydroxy-1,1-bisphosphonate+(EHBP)+on+endochondral+ossification+lesions+induced+by+a+lethal+oral+dose+of+uranyl+nitrate.">Effect of ethane-1- hydroxy-1,1-bisphosphonate (EHBP) on endochondral ossification lesions induced by a lethal oral dose of uranyl nitrate.</a> Arch. Toxicol. 79 (8), 475-481 (2005).</li><li>Kurttio, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+as+a+possible+target+of+chemical+toxicity+of+natural+uranium+in+drinking+water.">Bone as a possible target of chemical toxicity of natural uranium in drinking water.</a> Environ. Health Perspect. 113 (1), 68-72 (2005).</li><li>Collin-Osdoby, P., Osdoby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RANKL-mediated+osteoclast+formation+from+murine+RAW+264.7+cells.">RANKL-mediated osteoclast formation from murine RAW 264.7 cells.</a> Methods Mol. Biol. 816, 187-202 (2012).</li><li>Beranger, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+binding+of+poly(ADP-Ribose)+polymerase-1+and+JunD/Fra2+accounts+for+RANKL-induced+Tcirg1+gene+expression+during+osteoclastogenesis.">Differential binding of poly(ADP-Ribose) polymerase-1 and JunD/Fra2 accounts for RANKL-induced Tcirg1 gene expression during osteoclastogenesis.</a> J. Bone Miner. Res. 22 (7), 975-983 (2007).</li><li>Mirto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+uranium(VI)+speciation+for+the+evaluation+of+in+vitro+uranium+cytotoxicity+on+LLC-PK1+cells.">Influence of uranium(VI) speciation for the evaluation of in vitro uranium cytotoxicity on LLC-PK1 cells.</a> Hum. Exp. Toxicol. 18 (3), 180-187 (1999).</li><li>Carri&#232;re, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+uranium+speciation+on+normal+rat+kidney+(NRK-52E)+proximal+cell+cytotoxicity.">Influence of uranium speciation on normal rat kidney (NRK-52E) proximal cell cytotoxicity.</a> Chem. Res. Toxicol. 17 (3), 446-452 (2004).</li><li>Milgram, S., Carri&#232;re, M., Malaval, L., Gouget, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+accumulation+and+distribution+of+uranium+and+lead+in+osteoblastic+cells+as+a+function+of+their+speciation.">Cellular accumulation and distribution of uranium and lead in osteoblastic cells as a function of their speciation.</a> Toxicology. 252 (1-3), 26-32 (2008).</li><li>Milgram, S., Carri&#232;re, M., Thiebault, C., Malaval, L., Gouget, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+and+phenotypic+effects+of+uranium+and+lead+on+osteoblastic+cells+are+highly+dependent+on+metal+speciation.">Cytotoxic and phenotypic effects of uranium and lead on osteoblastic cells are highly dependent on metal speciation.</a> Toxicology. 250 (1), 62-69 (2008).</li><li>Studzinski, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Growth, Differentiation and Senescence A Practical Approach. , (1999).</li><li>Gritsaenko, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+uranium+impairs+the+differentiation+and+the+resorbing+function+of+osteoclasts.">Natural uranium impairs the differentiation and the resorbing function of osteoclasts.</a> Biochim Biophys Acta. 1861 (4), 715-726 (2017).</li><li>Motiur Rahman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation-coupled+osteoclast+differentiation+by+RANKL:+Cell+density+as+a+determinant+of+osteoclast+formation.">Proliferation-coupled osteoclast differentiation by RANKL: Cell density as a determinant of osteoclast formation.</a> Bone. 81, 392-399 (2015).</li></ol>

作者没有透露

据我们所知, 这是第一次详细的程序, 目的是研究天然铀对骨吸收细胞的影响。这种方法将有助于更好地了解铀对骨骼生理的影响, 并可能为铀合剂的筛选提供一个有趣的新工具。此外, 我们认为, 此处所述的议定书可用于研究其他重金属对 osteoclatogenesis 的影响。
众所周知, 铀在培养基中与无机和有机成分复合,18,19,<sup class="xr...

铀是一种天然存在于土壤、空气和水中的放射性元素;因此, 动物和人类在他们的饮食中接触到天然铀。除天然来源外, 铀来源于人为活动, 增加了其在环境中的丰度。铀造成化学和放射性危害。然而, 由于天然铀 (这是一种含有 99.27% 238u、0.72% 235u 和 0.006% 234u) 的同位素混合物, 具有低特定活动 (25.10 3. g-1), 其对健康的影响归因于其化学毒性。
无论其进入途径 (吸入、摄食或皮肤暴露), 大多数进入人体的铀都是用粪便清除的, 只有一小部分到达全身循环。大约67% 的铀在血液中依次被肾脏过滤, 并在 24 h1内将身体留在尿液中。其余部分大多沉积在肾脏和骨骼中, 两个主要靶器官的铀毒性2,3,4。由于骨架已被确定为铀长期保留期的主要站点2,3,4,5,6, 已进行了几项研究, 以探索铀对骨生理的影响7。
骨是一种矿化组织, 在其一生中不断被重塑。骨重塑是一个复杂的过程, 依赖于专门的细胞类型和主要包括两个阶段: 前已存在的旧基质的骨吸收, 然后再由成骨细胞构建。破骨细胞是由造血原细胞融合而成的大型多干细胞, 移植到它们附着在骨8的吸收部位。他们的附件同时发生与他们的细胞骨架的广泛的整顿9。这种重组需要建立在细胞和骨表面之间的隔离隔间, 其中的破骨体分泌质子, 导致羟基磷灰石的溶解, 以及参与降解的蛋白酶有机基质。由此产生的降解产物是 endocytosed 的, 通过细胞传送到与骨表面相对的膜区并分泌, 这一过程称为 transcytosis10,11。
结果从体内和体外研究表明, 铀抑制骨形成和改变成骨细胞的数量和活性7,12。相比之下, 铀对骨质吸收和破骨细胞的影响却没有得到很好的探索。几个体内研究报告了铀硝酸对小鼠或大鼠的骨吸收的增强作用13,14。此外, 一项流行病学调查表明, 通过饮水增加的铀摄入量往往与男子15的骨吸收标记物的血清水平增加有关。综上所述, 这些结果导致了这样的结论: 铀积累在骨骼中可以促进骨吸收。然而, 涉及铀的这种潜在作用的细胞机制仍然是一个有待商榷的问题。因此, 我们决定研究铀对吸收骨细胞行为的影响。
在这里, 我们描述的协议, 我们已经建立的特点和量化的影响, 天然铀的前破骨细胞的生存能力和破骨细胞分化和吸收活动。本文所述的实验已经完成了原始的264.7 小鼠转化巨噬细胞系, 这可以很容易地区分成骨细胞时, 培养在存在的因子 RANKL 4 或5天, 这是经典的研究破骨细胞分化与功能16。所开发的程序是可靠的, 给予高度重现性的结果, 完全适用于原发性破骨细胞。基于所有这些原因, 我们认为这种方法有助于更好地了解骨中铀毒性所涉及的分子机制。此外, 我们认为, 这一方法可作为筛选新的铀螯合剂的工具加以调整。

<table><tbody><tr><td>DMEM</td><td>Lonza</td><td>BE12-604F</td><td></td></tr><tr><td>&amp;alpha;-MEM</td><td>Lonza</td><td>BE12-169F</td><td></td></tr><tr><td>EMEM without phenol red</td><td>Lonza</td><td>12-668E</td><td></td></tr><tr><td>Water for cell culture</td><td>Lonza</td><td>BE17-724F</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Penicillin-Streptomycin solution</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>&amp;nbsp;L-Glutamine solution</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>Trypan Blue Solution 0.4%</td><td>Sigma-Aldrich</td><td>T8154</td><td></td></tr><tr><td>HyClone fetal bovine serum</td><td>GE Life Sciences</td><td>SH30071.03</td><td></td></tr><tr><td>7.5% sodium bicarbonate aqueous solution</td><td>Sigma-Aldrich</td><td>S8761</td><td></td></tr><tr><td>Acid Phosphatase, Lekocyte (TRAP) kit</td><td>Sigma-Aldrich</td><td>387A</td><td></td></tr><tr><td>Thiazolyl Blue Tetrazolium Bromide (MTT) powder</td><td>Sigma-Aldrich</td><td>M5655</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D5879</td><td></td></tr><tr><td>Alizarin Red S sodium salt, 1% w/v aq. sol.</td><td>Alfa Aeros</td><td>42746</td><td></td></tr><tr><td>Osteoassay bone resorption plates, 24 well plates</td><td>Corning Life Sciences</td><td>3987</td><td></td></tr><tr><td>Multiwell 24 well plates</td><td>Falcon</td><td>353504</td><td></td></tr><tr><td>Flask 75 cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Falcon</td><td>353133</td><td></td></tr><tr><td>Polypropylene Conical Tubes 50 ml</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Cell scrapers 30 cm</td><td>TPP</td><td>90003</td><td></td></tr></tbody></table>

methods for analyzing the impacts of natural uranium on in vitro osteoclastogenesis

铀已经被证明会干扰骨骼生理学, 并且它是公认的, 这种金属在骨骼中积累。然而, 对于天然铀对骨细胞行为的影响却知之甚少。特别是, 铀对破骨细胞的影响, 即负责骨质基质的吸收, 没有记录在案。为了研究这个问题, 我们已经建立了一个新的协议, 使用铀乙酸作为天然铀的来源和小鼠的原始264.7 细胞系作为破骨前体的模型。在这里, 我们详细介绍了测试铀对破骨细胞的毒性作用所需的所有化验结果, 并评估其对破的影响, 以及成熟破骨的吸收功能。我们所开发的条件, 特别是用于制备含铀的培养基和原始264.7 细胞的播种, 可以获得可靠和高度的生殖结果。此外, 我们还优化了使用软件工具, 以方便分析各种参数, 如破骨细胞的大小或吸收矩阵的百分比。

抗酒石酸酸性磷酸酶染色被用来可视化破骨细胞为具有3或更多核的大紫细胞核。在 RANKL 和铀离子的存在下培养的原始264.7 细胞获得的破骨组织的代表性图像如图 1所示。在整个水井和放大的图片中, 对铀的反应中, 破骨细胞的数量和大小的变化是很容易看到的。
使用 ImageJ 软件 (图 2...

Watch this Scientific Journal Video about Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis at JoVE.com

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

众所周知, 铀会影响骨骼的新陈代谢。在这里, 我们提出了一项协议, 目的是研究天然铀暴露对破骨细胞的活性、分化和功能的影响, 这是负责骨质吸收的单元。

天然铀对体外破影响的分析方法

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about ...

methods-for-analyzing-impacts-natural-uranium-on-vitro

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

8.1K 次观看.  Université Nice Sophia-Antipolis. 众所周知, 铀会影响骨骼的新陈代谢。在这里, 我们提出了一项协议, 目的是研究天然铀暴露对破骨细胞的活性、分化和功能的影响, 这是负责骨质吸收的单元。

视频: Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

去除微量元素的氧化铜纳米粉体制备铀<em&gt;原位</em&gt;恢复泌水及其对细胞活力的影响

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

科研

环境科学

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 ...

癌症研究

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

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

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 ...

发育生物学

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

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 ...

生物学

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.

大鼠骨髓破骨衣原体的分离、纯化及鉴别

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Resorption Pit Assay: An In Vitro Technique to Quantify Calcium Resorption Activity of Mature Osteoclasts using Calcium Phosphate-Coated Culture Plates

Source: Cen, W., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/64016">A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro.</a> J. Vis. Exp. (2022)
In this video, we describe an in vitro osteoclastic resorption assay on calcium phosphate-coated cell culture plates cultured with human peripheral blood mononuclear cell-derived osteoclasts. The osteoclasts form a characteristic actin ring on the calcium phosphate-coated matrix of the cell culture plate and degrade the matrix within the actin ring region to release calcium and phosphate ions, resulting in the formation of distinct resorption pits within the degraded matrix.

吸收坑测定:一种使用磷酸钙包被培养板定量成熟破骨细胞钙吸收活性的体外技术

Source: Cen, W., et al. A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro. J. Vis. Exp. (2022)
In this video, we ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Cell-based assays

Effect of the Anti C-fms Antibody on Osteoclastogenesis in Mouse Bone Marrow Cells In Vitro

Source: Marahleh, A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59089">Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the effect of different anti-c-fms antibody concentrations on osteoclastogenesis, which is the process of osteoclast formation from osteoclast progenitors.

抗 C-fms 抗体对小鼠骨髓细胞破骨细胞体外生成的影响

Source: Marahleh, A., et al. Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro. J. Vis. Exp. 
...

天然铀对体外破影响的分析方法

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去除微量元素的氧化铜纳米粉体制备铀