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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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. 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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. 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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,18,19,20,21。这些 complexations 影响铀的形态, 并以这种方式, 它的细胞毒性。因此, 该议定书的一个关键步骤是准备含铀的培养基。我们以前已经显示了23 , 在原始264.7 细胞培养基中存在5% 胎牛血清对铀的细胞毒性没有显著影响, 使用浓度范围从0到400µM。这是一个重要的问题, 因为在培养基中血清的存在需要分析铀在整个破分化过程中的暴露作用。然而, 我们要强调的是, 为准备曝光介质 (6 小时) 所描述的漫长的两步程序必须仔细地遵循。事实上, 在每次稀释铀盐3小时后的孵化步骤允许在进一步稀释或细胞暴露之前在溶液中可能形成的铀配合物的稳定。这是绝对需要获得可靠和重现性的结果。
另外一个重要的参数的重复性破骨细胞分化和吸收化验是播种密度的原始 264. 7 单元格。事实上, 它已经表明, 单核前体密度是一个关键的决定因素, 破骨细胞的形成, 很可能是因为单元-细胞接近影响细胞融合事件24。因此, 必须特别注意细胞计数, 细胞悬浮准备和播种的均匀性在每个井, 为了避免误解。
该阈值工具是有用的自动量化的吸收。值得指出的是, 这种分析需要正确的照明图像。然而, 无论是相机的类型还是图像采集方法, 一个常见的问题是图像边缘的光照不均匀。在这种情况下, 可能需要多步阈值法。
总而言之, 我们已经建立了一个健壮的协议, 允许研究铀对破骨细胞的形成、活性和功能的影响。本程序是利用原始的264.7 细胞线, 但完全适用于原发骨髓破骨的前体, 我们已经显示23。

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>&#945;-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>&#160;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 cm2</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) 分析破骨细胞的大小。为此, 使用了陷阱染色破骨细胞的全井图像, 并在每个培养条件 (图 2A和 2D) 的每个井中处理了相同的区域。所有的破骨细胞存在于每个区域中 ( 图 2B和2E), 这些都允许确定其区域 (以µm2) ( 图 2C和2F表示)。如图 2所示的示例说明了铀对破骨细胞大小的强烈影响。
为了研究铀对破骨细胞吸收活性的影响, 在 osteomimetic 表面板上镀并分化了264.7 个原电池。在化验结束时, 破骨细胞被移除。然后, 以茜素红 S 钠盐处理各井中的骨模拟表面, 获得了吸收区的染色, 得到了每一个井的图像 (图 3A和 3D)。生成的图片是用图像 J 软件处理的。它们在8位灰度图像 (图 3B和 3E) 中被转换, 受到阈值 ( 数字 3C和3F) 和吸收区域 (白色区域) 的自动计算。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56499/56499fig1.jpg"> 图 1: U (VI) 对破骨细胞形成的影响的可视化.未加工的264.7 细胞在被表明的集中的存在被区分了铀。4天, 抗酒石酸酸性磷酸酶 (TRAP) 染色进行。代表性全井显微 (上部板) 显示在这些条件下获得的陷阱染色破骨细胞。核破骨细胞在盒装区域的例子显示在一个更高的放大倍数 (箭头在底部面板)。这些图片说明了 U (VI) 对破骨细胞形成的剂量依赖性效应。黑头箭头表示破骨细胞核的例子。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56499/56499fig1large.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/56499/56499fig2.jpg"> 图 2: 分析 U (VI) 对破骨细胞大小的影响.(A和D): 对破骨细胞的全井图像进行了分析, 并给出了相应的区域。(a 和 D) 中的红色方框分别对应于 (B) 和 (E) 中所示的区域。(B和E): 两个铀浓度条件下的破骨细胞边缘的手工绘制以蓝色显示。(C和F): 来自 ImageJ 软件的结果窗口分别显示 (B 和 E) 分析的数据。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56499/56499fig2large.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/56499/56499fig3.jpg"> 图 3: 分析 U (VI) 对破骨细胞功能的影响.在缺席 (A) 或存在25µM 的铀 (D) 后, osteomimetic 表面的茜素染色后的图像破吸收发生。在 (A和D) 中的图片首先在8位灰度图像中进行了转换, 如下所示 (B和E), 然后在二进制图像 (C和F) 中应用适当的阈值设置。这些二进制图像用于计算每个条件中的面积吸收的百分比。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56499/56499fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

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

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

生产和使用慢病毒的有选择性地转导原少突胶质前体细胞<em&gt;在体外</em&gt;髓鞘测定

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

科研

发育生物学

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the &#8216;&#8216;stemness&#8217;&#8217; of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia.

协议中再生的小鼠肠道使用条件潘氏细胞中的作用分析<em&gt;的Cre-LOX</em&gt;小鼠模型

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

方法来检查淋巴腺和血细胞中<em&gt;果蝇</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

活体果蝇卵巢组织中卵泡干细胞谱系的细胞内 pH 值的成像方法

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

rna 干扰和热休克诱导转录因子表达在重编程生殖细胞到神经元中的应用C. 线虫

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

Stentor再生的研究方法

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the formation of new blood vessels and involve several signal transduction pathways. Here we show that the tube formation assay is a simple in vitro method to evaluate the impact of natural products on angiogenesis and to investigate the molecular mechanisms involved. In particular, in the presence of the water extract of Ruta graveolens (RGWE), endothelial cells are no longer able to form a cell-cell network and that the RGWE effects on human umbilical vein endothelial cell (HUVEC) tube formation is abolished by the constitutive activation of MEK.

Here, we evaluate the effects of the water extract of Ruta graveolens on vessel network formation by using a tube formation assay on a gelled basement matrix.

HUVEC 管形成测定评估天然产物对血管生成的影响

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-&#945;-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed.

Methods to Test Endocrine Disruption in Drosophila melanogaster

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

检测果蝇黑色素的内分泌干扰的方法

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

分离大鼠脂肪组织间充质干细胞以分化为产生胰岛素的细胞

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

天然铀对体外破影响的分析方法

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生产和使用慢病毒的有选择性地转导原少突胶质前体细胞