这项工作是由一个 Plaxgen 研究补助金授予 SM (PLX-1008)。我们感谢帕洛阿尔托医学研究基金会研究机构在 IRB 的批准下收集动脉粥样硬化患者的血清样本。

1. 荧光标记的胆固醇和降脂药物的制备 注: 荧光标记的胆固醇聚集物和他汀类制剂, 如我们前文所述 21 . 有关试剂、成像流式细胞仪、化学分析仪和本研究中使用的数据分析软件的详细信息, 请参阅 材料表 。
<ol> <li> 溶解荧光标记的冻干胆固醇 (1 毫克, 前/Em = 495 nm/507 纳米) 粉末在1毫升100% 酒精. </li>
	<li> 离心样品3分钟在 2040 x g, 并使用清含有可溶性荧光标记的胆固醇聚集在斑块阵列的检测. </li>
	<li> 用于药物的制备, 分别溶解辛伐他汀、洛伐他汀、阿托伐他替和 omega-3 脂肪酸的粉末 (2 毫克), 1 毫升100% 乙醇. </li>
	<li> 在离心后3分钟在 2040 x g, 使用清含有药物的化验. </li>
	<li> 同样地, 溶解在1毫升的去离子水中单独地将氟伐他汀、他、烟酸和贝特的粉末 (2 毫克) 制作成2毫克/毫升的股票溶液. </li>
	<li> 在 2040 x g 离心3分钟后, 在斑块检测中使用含有药物的清. </li>
</ol> 2。斑块阵列法检测 体外 胆固醇颗粒形成 <ol> <li> 使用化学 analyzer-1 (见材料表) 建立斑块阵列法测定胆固醇颗粒的形成。使用1节所制备的试剂, 在圆底、低蛋白结合96好的板上进行化验。在200和 #181 中保持每一个井的最终反应量; L 和执行所有的化验三份. 
	<ol> <li> 首先, 将194.5 和 #181 的磷酸盐缓冲盐水 (PBS) 放到每个井中. </li>
		<li> 每一个井, 添加2.5 和 #181; L (5 和 #181; g) 每个降脂药物溶液和震动板三十年代通过放置在化学 analyzer-1 反应板, 以均匀混合药物成溶液。阴性对照样品中不添加任何药物. </li>
		<li> 最后, 添加2和 #181; L (2 和 #181; g) 用荧光标记的胆固醇骨料溶液对每一个井, 并动摇板三十年代通过放置在化学 analyzer-1 反应板. </li>
		<li> 在实验室的振动筛上孵育2小时37和 #176; C 和 200 rpm. </li>
		<li> 孵化后, 通过成像流式细胞仪获取颗粒图像. </li>
	</ol> </li> </ol> 3。利用成像流式细胞仪采集胆固醇颗粒图像 <ol> <li> 打开数据获取模板以加载正确的仪器设置。单击和 #34; 文件和 #34;, 然后选择和 #34; 打开模板和 #8230; #34; 选择模板文件. </li>
	<li> 单击和 #34; 冲洗、锁定、加载和 #34; 准备样品装载的仪器. </li>
	<li> 当和 #34; 加载示例和 #34; 对话框打开, 单击并 #34; 确定和 #34; 加载50和 #181; 在2节中的每一个井中准备的样品进入成像流式细胞仪. </li>
	<li> 单击并 #34; 运行 |设置和 #34; 实时查看成像区域中的粒子. </li>
	<li> 当成像区域中的粒子处于良好焦点时, 单击并 #34; 运行 |获取和 #34; 以高通量的方式同时获取每个对象的图像 (侧面散射/SSC)、亮场 (BF)、绿色荧光和黄色荧光. </li>
	<li> 重复步骤 3.2-3.5, 从每个样本中获取 5000-1万粒子. </li>
	<li> 使用图像分析软件分析所有原始图像文件, 以确定对象的荧光强度和形态学变化. 
	<ol> <li> 单击 "#34; 工具和 #34; 在图像分析软件顶部的按钮, 然后选择和 #34; 批处理数据文件和 #34; 从下拉菜单中, 将打开和 #34; 批次和 #34; 窗口. </li>
		<li> 在 #34; 批处理和 #34; 窗口中, 单击并 #34; 添加批处理和 #34; 按钮, 它将打开和 #34;D efine 批次和 #34; 窗口. </li>
		<li> #34;D efine 批处理 #34; 窗口中, 单击和 #34; 添加文件和 #34; 按钮并选择原始图像文件, 单击 #34; 模板和 #34; 按钮, 然后选择相应的数据分析模板文件, 然后选择和 #34; 确定和 #34; #34; 提交批次和 #34;开始处理和分析原始图像文件. </li>
	</ol> </li> </ol> 4。基于斑块阵列法的降脂药物对纯化胆固醇颗粒影响的评价 <ol> <li> 如步骤2.1 所述, 使用纯化的脂蛋白、LDL 和高密度脂蛋白/微粒来进行胆固醇颗粒的形成试验。在96井板上进行斑块阵列检测. 
	<ol> <li> 首先, 用 PBS 加载所有井; 使用200和 #181; 每个井的最终音量. </li>
	</ol> </li> <li> 用于负控制, 添加4和 #181; g 脂蛋白、LDL 或高密度脂蛋白蛋白/微粒单独地在每个井没有药物并且震动板材三十年代. 
	<ol> <li> 用于检查降脂药物的效果, 添加4和 #181; g 脂蛋白, LDL, 或高密度脂蛋白蛋白/颗粒单独到每一个井和动摇板三十年代. </li>
		<li> 为4和 #181 添加的水井; g 脂蛋白, LDL, 或高密度脂蛋白, 增加2.5 和 #181; L (5 和 #181; g) 替, 洛伐他汀, 辛伐他丁, 或烟酸溶液, 并动摇板三十年代通过放置在化学 analyzer-1. </li>
		<li> 最后, 添加2和 #181; L (2 和 #181; g) 荧光标记的胆固醇聚合溶液, 所有水井和动摇板三十年代通过放置在化学 analyzer-1 反应板. </li>
	</ol> </li> <li> 在37和 #176 的实验室振动台中孵育2小时的板; C 和 200 rpm. </li>
	<li> 在步骤 3.1-3.5 中使用成像流式细胞仪获取样本. </li>
	<li> 如步骤3.7 所述, 使用图像分析软件分析原始图像文件. </li>
	<li> 在分析模板中, 使用以下浇口方案来识别胆固醇粒子亚群. 
	<ol> <li> 在绿色通道饱和像素计数 (x 轴) 和暗域通道饱和像素计数 (y-axis) 的绘图上, 在任一通道中拒绝具有一个或多个饱和像素的对象. </li>
		<li> 使用绿色通道强度 (x 轴) 和 ssc 强度 (y-axis) 绘制没有饱和像素的对象/粒子: 根据其绿色通道和 ssc 强度, 对象属于三个区域 (脂蛋白、LDL、高密度脂蛋白). 
		注意: 这些门是根据通过使用纯化的脂蛋白、LDL 和高密度脂蛋白粒子/蛋白质的控制实验所产生的数据而建立的. </li>
	</ol> </li> <li> 以确定每个样本的降脂药物效果, 计算总脂蛋白颗粒浓度, 以及步骤6.5 中描述的每个亚群 (脂蛋白、LDL、高密度脂蛋白) 的百分比. 
	<ol> <li> 使用以下公式计算粒子浓度 (粒子/mL): <img alt="Equation" src="/files/ftp_upload/56596/56596eq1.jpg"/> 注意: 脂蛋白、LDL 和高密度脂蛋白浇注区在荧光点-情节检测 ~80% 他们各自的亚人口, 并且剩余的20% 微粒与其他门控区重叠. </li>
	</ol> </li> </ol> 5。血清样品中脂质浓度的测定 <ol> <li> 用于通过化学 analyzer-2 测定 LDL、高密度脂蛋白、胆固醇和甘油三酯的浓度, 使用与年龄匹配的正常和血脂紊乱患者获得的血清样本. </li>
	<li> 定义 LDL、高密度脂蛋白、胆固醇和甘油三酯的异常浓度, 其依据是血清样本中的上、下参考值之外的浓度. </li>
	<li> 遵循正常参考值建议ded 由试剂制造商: 胆固醇 (4-200 毫克/dl), 高密度脂蛋白 (3.0-65 毫克/dl), LDL (4-100 毫克/dl), 和甘油三酯 (9-200 毫克/dl). </li>
	<li> 将具有异常值的血清样本识别为低于较低的参考值或高于胆固醇和甘油三酯的较高参考值, 并在存在和无降脂药物的情况下使用它们进行筛选. </li>
</ol> 6。药物对血清样品中胆固醇颗粒形成的影响分析 <ol> <li> 使用化学 analyzer-1 建立血清样品中胆固醇颗粒形成的斑块阵列法。在一个圆底, 低蛋白结合 96-井板进行化验。使用200和 #181 的最终反应量; L/良好, 并执行所有的化验三份. </li>
	<li> 通过逐步地加载试剂来准备板材. 
	<ol> <li> 准备控制井. </li>
		<li> 加载193和 #181; PBS 的每个井. </li>
		<li> 添加 2.5% (v/v) 患者血清 (每井仅一个血清样本), 并将其放在化学分析仪反应板上, 在三十年代摇动钢板. </li>
		<li> 添加2和 #181; L (2 和 #181; g) 将荧光标记的胆固醇聚合溶液放入每一个井中, 并将其放在化学 analyzer-1 反应板上, 使其在三十年代震动板块. </li>
	</ol> </li> <li> 使用药物准备水井. 
	<ol> <li> 加载191和 #181; PBS 对每个井. </li>
		<li> 添加 2.5% (v/v) 患者血清 (每井只有一个血清样本), 并将其放在化学 analyzer-1 反应板上, 将其摇动到三十年代. </li>
		<li> 添加2和 #181; 替、洛伐他汀或烟酸溶液 (4 和 #181; g) 除负控井外, 全部井。每井只添加一种药物, 并将其放在化学 analyzer-1 反应板上, 从而在三十年代摇动盘. </li>
		<li> 添加2和 #181; 荧光标记的胆固醇骨料溶液 (2 和 #181; g) 进入每一个井和动摇板三十年代通过放置在化学 analyzer-1 反应板. </li>
	</ol> </li> <li> 仅在所有水井 (控制和药物治疗) 加载了其各自的血清样本后才加载药物, 并在本步骤末尾添加荧光标记的胆固醇. </li>
	<li> 在37和 #176 的实验室振动台中孵育2小时的板; C 和 200 rpm. </li>
	<li> 在步骤 3.1-3.6 中描述的设置后, 通过成像流式细胞仪获取样本. </li>
	<li> 使用图像分析软件对所有图像文件进行批处理, 使用与步骤3.7 中所述相同的模板. 
	<ol> <li> 执行数据分析, 方法是在步骤4.5 和4.6 中所述的绘图上绘制门. </li>
	</ol> </li> <li> 用于确定每个样本的降脂药物效果/反应 (低、中、高), 计算总脂蛋白颗粒浓度, 以及包含脂蛋白、LDL 和高密度脂蛋白的总粒子的百分比亚人口. </li>
	<li> 绘制脂蛋白, 低密度脂蛋白和高密度脂蛋白在物体和 #39 的直方图上的分布, 从长度 (长度-宽度) 和门到球状或线性区域中减去的宽度 (长度-宽度) 和 #8804; 2 和 #160; #181; 我落入球状区域, 用于计算球状和线性形状粒子的百分比. </li>
	<li> 比较每种药物培养的血清样品中球状和线性型 LDL 和 HDL 胆固醇颗粒的百分比。在每个血清样本中获得的三个值中, 接受在 SD 和 #177 中的球状和线性型 LDL 和高密度脂蛋白粒子的百分比; 2 范围为药物作用的决心. </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=Clinical+relevance+of+the+biochemical,+metabolic,+and+genetic+factors+that+influence+low-density+lipoprotein+heterogeneity.">Clinical relevance of the biochemical, metabolic, and genetic factors that influence low-density lipoprotein heterogeneity.</a> Am J Cardiol. 90, 30-47 (2002).</li><li>Weissglas-Volkov, D., Pajukanta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+causes+of+high+and+low+serum+HDL-cholesterol.">Genetic causes of high and low serum HDL-cholesterol.</a> J Lipid Res. 51, 2032-2057 (2010).</li><li>Abela, G. S. <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+statins+on+cholesterol+crystallization+and+atherosclerotic+plaque+stabilization.">Effect of statins on cholesterol crystallization and atherosclerotic plaque stabilization.</a> Am J Cardiol. 107, 1710-1717 (2011).</li><li>Nidorf, S. M., Eikelboom, J. W., Thompson, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+cholesterol+crystal-induced+inflammation+for+the+secondary+prevention+of+cardiovascular+disease.">Targeting cholesterol crystal-induced inflammation for the secondary prevention of cardiovascular disease.</a> Cardiovasc Pharmacol Ther. 19, 45-52 (2014).</li><li>Thacker, S. G., Zarzour, A., Chen, , 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=High+density+lipoprotein+reduces+inflammation+from+cholesterol+crystals+by+inhibiting+inflammasome+activation.">High density lipoprotein reduces inflammation from cholesterol crystals by inhibiting inflammasome activation.</a> Immunol. 149, 306-319 (2016).</li><li>Kim, S. H., Lee, E. S., Lee, J. Y., 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=Multiplex+coherent+anti-stokes+Raman+Spectroscopy+images+intact+atheromatous+lesions+and+concomitantly+identifies+distinct+chemical+profiles+of+atherosclerotic+lipids.">Multiplex coherent anti-stokes Raman Spectroscopy images intact atheromatous lesions and concomitantly identifies distinct chemical profiles of atherosclerotic lipids.</a> Circ Res. 106, 1332-1341 (2010).</li><li>Lim, R. S., Suhalim, J. L., Miyazaki-Anzai, S., 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=Identification+of+cholesterol+crystals+in+plaques+of+atherosclerotic+mice+using+hyperspectral+CARS+imaging.">Identification of cholesterol crystals in plaques of atherosclerotic mice using hyperspectral CARS imaging.</a> J Lipid Res. 52, 2177-2186 (2011).</li><li>Rothblat, G. H., Phillips, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-density+lipoprotein+heterogeneity+and+function+in+reverse+cholesterol+transport.">High-density lipoprotein heterogeneity and function in reverse cholesterol transport.</a> Curr Opin Lipidol. 21, 229-238 (2011).</li><li>Kontush, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDL+particle+number+and+size+as+predictors+of+cardiovascular+disease.">HDL particle number and size as predictors of cardiovascular disease.</a> Front in Pharmacol. , (2015).</li><li>Karathanasis, S. K., Freeman, L. A., Gordon, S. M., Remaley, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+changing+face+of+HDL+and+the+best+way+to+measure+it.">The changing face of HDL and the best way to measure it.</a> Clin. Chem. 63, 196-210 (2017).</li></ol>

Dr. Madasamy 获得了 Plaxgen 公司的赠款支持, 并具有竞争性的财务利益。其他作者没有任何竞争的金融利益透露。

一般而言, 血液循环中脂蛋白、LDL 和高密度脂蛋白胆固醇颗粒的分布和功能特性主要由代谢、遗传、流行病学、细胞和血浆因素决定22,23。在本研究中, 观察脂质修饰药物在缓冲液中的作用, 发现高脂溶性药物如替、洛伐他汀、辛伐他丁、阿托伐他汀等对胆固醇颗粒的形态具有较高的复杂性。与高亲水性他和氟伐他汀药物的低水平效果比较。这些结果与我们以前的研究很好地一致, 描述了酶机制的作用, 他汀类药物在调节 LDL 和高密度脂蛋白胆固醇颗粒形成的缓冲和血清样本21。因此, 本研究的结果揭示了替、烟酸、贝特和 omega-3 脂肪酸药物的酶作用机制, 可能在调节胆固醇颗粒的形成中起直接影响。药物和胆固醇聚合体之间的相互作用可能导致大尺寸胆固醇颗粒的组装, 这些微粒是 2-60 µm2, 表现出球状和线性的链状形态。
此外, 使用纯化的脂蛋白粒子的结果表明, 胆固醇聚集物与血浆因子之间的相互作用, 包括脂蛋白、LDL 和高密度脂蛋白蛋白, 可能改变胆固醇的组成和形态特征。粒子.与纯化的脂蛋白微粒有关的药物治疗结果表明, 脂蛋白颗粒形成的药物效应与其对 LDL 胆固醇颗粒形成的效果相比有较高的水平。以洛伐他汀、辛伐他丁和替药物作为 pro-drugs, 其在测定中的用量可能高于生理浓度。
有趣的是, 血清样本的筛选显示, 药物对改变脂蛋白、ldl 和高密度脂蛋白胆固醇颗粒形成的影响不同, 特别是对线性型 ldl 和高密度脂蛋白粒子的影响。这些药物诱导减少的线性型 LDL 和高密度脂蛋白胆固醇颗粒形成的血脂异常和年龄匹配的正常血清样本。在辛伐他汀、替、洛伐他汀和烟酸中, 对减少线状颗粒形成的药物效应较高。在正常和血脂异常血清样品中, 用球状和线性链状形态识别胆固醇颗粒, 表明具有相似形貌的颗粒可能在体内条件下形成。先前的研究已经确定了在人的动脉粥样硬化斑块和载脂蛋白- -和 LDLR-小鼠模型24,25,26的存在椎间盘和针状胆固醇晶体,27,28。
血液中的高密度脂蛋白粒子作为一种非均质混合物存在, 且体积小、体积大的高密度脂蛋白粒子随着功能活动的进行, 是通过逆向胆固醇转运发挥其心保护作用的重要因素路径29,30。最近的研究强调了确定高密度脂蛋白胆固醇颗粒亚的重要性, 以阐明它们在多种生物功能中的作用, 如胆固醇外流、抗炎、血栓和抗氧化性31.此外, 一些研究报告了降脂治疗在血浆中增加低至中度高密度脂蛋白的作用1,5,21。因此, 本研究的结果为胆固醇颗粒的形态特征提供了新的见解。值得注意的是, 在血脂异常患者的血清样本中检测到更高水平的线性形状的高密度脂蛋白胆固醇颗粒, 表明它们可能是诊断和评价脂质修饰药物的可靠的生物标志物。然而, 需要进一步的调查, 使用大型临床标本, 以更好地了解不同形态的胆固醇颗粒和它们的关联与 CVD。
在检测药物对胆固醇颗粒组装的影响的斑块检测中, 我们使用了2µg 的荧光标记的胆固醇总量和5µgof 每种药物, 因为: (1) 药物具有竞争性的结合荧光标记的胆固醇和血清样品中的内源性脂质;(2) 从每个样本, 我们获得5000到1万的胆固醇微粒组装成大尺寸和形状范围从〜 2-60 µ2;(3) 我们观察到与药物一起孵育的血清样本 (剂量 300 ng 至5µg) 和 ~ 1-5% 的药物反应在高剂量孵育的情况下有很大的变化, 在胆固醇颗粒形成的剖面上没有发现可检测的变化;(4) 胆固醇聚集物与降脂药物的相互作用由酶过程介导。因此, 测定试剂的浓度可能高于其生理水平。
总之, 我们已经成功地证明了一个体外成像方法的优点, 这项研究中描述了广泛的降脂药物对胆固醇的调节形态和组成的影响。粒子.利用图像分析算法对脂质颗粒的形态进行可视化和定量化的方法, 可能有助于动脉粥样硬化的诊断和评价降脂治疗的效果。

大量的临床研究表明, 降脂药物在降低低密度脂蛋白胆固醇的血浆水平和高密度脂蛋白 (HDL) 胆固醇的低水平到中等程度的增加中有有益的作用, 这防止动脉粥样硬化相关不良心血管事件的主要和第二次发生1,2,3,4,5。他汀类, 一组的英国女王陛下的还原酶抑制剂, 阻断内源性胆固醇合成在肝脏, 反过来导致低密度脂蛋白胆固醇在血液中的循环水平6,7。同样, 烟酸的降脂作用是介导其直接和非抑制肝细胞甘油 acyltransferase-2, 一个关键的肝酶参与甘油三酯合成8。相比之下, 替通过限制外源性胆固醇的吸收, 通过在小肠上皮细胞中的尼曼 C1-Like 1 (NPC1L1) 蛋白的结合来降低血浆 LDL 的水平.9。非诺贝特, 另一个降脂药物, 大大降低了血浆甘油三酯的浓度, 也适度降低 LDL 胆固醇通过过氧化物扩散激活受体通路10。此外, omega-3 脂肪酸报告有抗的影响, 因为它的能力, 以降低血浆 LDL 水平的低密度脂蛋白11。
降脂药物除了对降低 LDL 胆固醇的主要作用外, 还有许多有益的效效应, 包括提高 HDL 水平, 改善内皮功能, 减少炎症, 抑制血小板聚合12,13,14。然而, 这些药物在增加高密度脂蛋白胆固醇颗粒和改变其结构特征方面的基本机制还没有得到充分的了解。由于这些药物被广泛用于治疗动脉粥样硬化相关的心血管疾病 (心血管), 因此必须进一步研究它们在确定脂质颗粒的形态特征和分布方面可能发挥的作用。人血浆 lipidome 包括大约600不同的脂质和22各种各样的分子类型胆固醇存在于各种各样的大小、形状、密度和构成15,16,17.分析方法, 如 ultra-centrifugation, 核磁共振, 梯度凝胶电泳用于表征 LDL 和高密度脂蛋白粒子及其亚18,19。然而, 这些方法的应用仅限于研究药物在调节脂质颗粒的形态和组装中的作用。流式细胞仪基斑块阵列是一种功能生化检测和可视化血清衍生脂质和淀粉样菌斑块颗粒20。本研究中所述的体外成像方法的优点, 可在缓冲液和血清样品中改变胆固醇颗粒的形态和分布, 从而识别出脂质调节药物的作用。

<table>
	<tbody>
		<tr>
			<td>TopFluor fluorescent cholesterol</td>
			<td>Avanti Polar lipids</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>simvastatin (pro-drug)</td>
			<td>Cayman Chemicals</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>lovastatin (pro-drug)</td>
			<td>Cayman Chemicals</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>rosuvastatin</td>
			<td>Cayman Chemicals</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>atorvastatin</td>
			<td>Cayman Chemicals</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>fluvastatin</td>
			<td>Cayman Chemicals</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>ezetimibe (pro-drug)</td>
			<td>Cayman Chemicals</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Niacin</td>
			<td>MilliporeSigma</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>fibrate</td>
			<td>MilliporeSigma</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>omega-3 fatty acid</td>
			<td>MilliporeSigma</td>
			<td></td>
			<td>store 100 &#181;l aliquots at -20 &#176;C</td>
		</tr>
		<tr>
			<td>purified VLDL proteins/particles</td>
			<td>Lee Bio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>purified LDL proteins/particles</td>
			<td>Lee Bio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>purified HDL proteins/particles</td>
			<td>Lee Bio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human age-matched serum</td>
			<td>Dx Biosamples</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human atherosclerosis serum</td>
			<td>Bioserve</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human normal serum</td>
			<td>Stanford Blood center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LDL measurement reagent pack</td>
			<td>Roche Diangostics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HDL measurement reagent pack</td>
			<td>Roche Diangostics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Total cholesterol measurment</td>
			<td>Roche Diangostics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well microtitre plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triglycerides measrument</td>
			<td>Roche Diangostics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amnis Imaging Flow cytometer</td>
			<td>Amnis Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IDEAS image analysing software</td>
			<td>Amnis Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemistry Analyzer-1, ChemWel 2902</td>
			<td>Awarness Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemistry Analyzer-2, Intergra 400</td>
			<td>Roche Diangostics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

differential effects of lipid-lowering drugs in modulating morphology of cholesterol particles

治疗血脂异常患者降脂药物导致低密度脂蛋白 (LDL) 水平的显著降低, 并在血浆高密度脂蛋白胆固醇 (HDL) 中降低到中等程度的增加。然而, 这些药物在改变胆固醇颗粒的形态和分布方面可能发挥的作用是不清楚的。在这里, 我们描述了体外应用斑块阵列法结合成像流式细胞仪对胆固醇颗粒的形态特征进行降血脂作用的评价。对胆固醇微粒的图像分析表明, 洛伐他汀、辛伐他丁、替和阿托伐他汀诱导球状和线形颗粒的形成, 而烟酸、类、氟伐他汀和他诱导只形成球状形状的微粒。其次, 纯化的非常低密度脂蛋白 (脂蛋白) 和 LDL 粒子与这些药物的培养表明, 胆固醇微粒亚群的形态学和图像纹理的变化。此外, 对50血清样本的筛选显示, 与年龄匹配的正常 (平均为 11.1%) 样本相比, 血脂异常 (平均为 18.3%) 的患者存在较高的线性型高密度脂蛋白胆固醇颗粒。我们还观察到, 降血脂药物对减少血清样品中的线性型 LDL 和高密度脂蛋白胆固醇颗粒的影响有相当大的变化。这些结果表明, 降脂药物, 除了其细胞介导的降效应, 可以直接调节胆固醇颗粒的形态的酶机制的行动。这些结果有可能通知动脉粥样硬化的诊断和预测最佳降脂治疗。

Watch this Scientific Journal Video about Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles at JoVE.com

降脂药物对胆固醇颗粒形态调节的不同作用医学|视频

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

降血脂药物对胆固醇颗粒形态的差异化作用

本研究的目的是评价体外降血脂药物对胆固醇颗粒形态的调节作用。对降血脂药物的比较表明, 它们在调节胆固醇颗粒的形态特征上有不同的作用。

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to ...

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Medicine

Fundamentals

Chemistry of Life

SCIENTISTS IN ACTION

14.5K 次观看.  Plaxgen Inc. 本研究的目的是评价体外降血脂药物对胆固醇颗粒形态的调节作用。对降血脂药物的比较表明, 它们在调节胆固醇颗粒的形态特征上有不同的作用。

视频: Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

Cholesterol Efflux Assay

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2. Excessive cholesterol is more common problem 3. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. 
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.

The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.

胆固醇外流检测

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

科研

医学

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

方法论方法血管功能和形态的非侵入性评估

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis.  Damage to the endothelium may ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

人类成胶质细胞瘤器官型切片培养模型，用于研究肿瘤细胞迁移和抗创伤药物的患者特异性作用

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

评估抗癌药物的特异性<em&gt;体外</em

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. Several drugs are administered into the anterior chamber or vitreous, and the intraocular injection has been effective in curing various intraocular diseases. Rabbit eyes have been widely used for ophthalmic research, as the animal is easy to handle and economical compared to other mammals, and the size of a rabbit eye is similar to that of a human eye. Using a 30 G needle, drugs can be injected into the intracameral and intravitreal spaces of rabbit eyes. The eyeballs are then frozen until analysis, and can be divided into the aqueous humor, vitreous, and retina/choroid. The vitreous and retina/choroid samples can be homogenized and solubilized before analysis. Then, immunoassays can be performed to measure the concentrations of intraocular drugs in each compartment. Appropriate pharmacokinetic models can be used to calculate several parameters, such as the half-life and maximum concentration of the drug. Rabbit eyes can be a good model for pharmacokinetic studies of intraocular drugs.

Rabbits are widely used to study the pharmacokinetics of intraocular drugs. We describe a method for conducting pharmacokinetic studies of intraocular drugs using rabbit eyes.

眼内药物的药代动力学研究采用兔眼

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

超声心动图与小鼠心脏形态学的组织学检查

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

小鼠骨骼肌形态及纤维型成分的半自动化分析

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

高通量硝基苯并二氮唑标记的胆固醇积液测定

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in ...

Self-Administration of Drugs in Mouse Models of Feeding and Obesity

Preclinical studies in mice often rely on invasive protocols, such as injections or oral gavage, to deliver drugs. These stressful routes of administration have significant effects on important metabolic parameters including food intake and body weight. Although an attractive option to circumvent this is to compound the drug in rodent food or dissolve it in water, these approaches also have limitations as they are affected by drug stability at room temperature for extended periods of time, the drug's solubility in water, and that the dosing is highly dependent on timing of food or water intake. The constant availability of the drug also limits translational relevance on how drugs are administered to patients. To overcome these limitations, drugs can be mixed with highly palatable food, such as peanut butter, allowing mice to self-administer compounds. Mice reliably and reproducibly consume the drug/peanut butter pellet in a short time frame. This approach facilitates a delivery approach with minimal stress compared with an injection or gavage. This protocol demonstrates the approach of drug preparation, animal acclimatization to placebo delivery, and drug delivery. The implications of this approach are discussed in studies related to timing of drug administration and the circadian rhythm.

The overall goal of this procedure is to describe a method for self-administration of drugs that can be used in mouse models of feeding and obesity.

喂养和肥胖小鼠模型中的药物自我管理

Preclinical studies in mice often rely on invasive protocols, such as injections or oral gavage, to deliver drugs. These stressful routes of ...

Rectal Organoid Morphology Analysis (ROMA): A Diagnostic Assay in Cystic Fibrosis

Diagnosis of cystic fibrosis (CF) is not always straightforward, especially when sweat chloride concentration is intermediate and/or less than two disease-causing CFTR mutations can be identified. Physiological CFTR assays (nasal potential difference, intestinal current measurement) have been included in the diagnostic algorithm but are not always readily available or feasible (e.g., in infants). Rectal organoids are 3D structures that grow from stem cells isolated from crypts of a rectal biopsy when cultured under specific conditions. Organoids from non-CF subjects have a round shape and a fluid-filled lumen, as CFTR-mediated chloride transport drives water into the lumen. Organoids with defective CFTR function do not swell, retaining an irregular shape and having no visible lumen. Differences in morphology between CF and non-CF organoids are quantified in the &#39;Rectal Organoid Morphology Analysis&#39; (ROMA) as a novel CFTR physiological assay. For the ROMA assay, organoids are plated in 96-well plates, stained with calcein, and imaged in a confocal microscope. Morphological differences are quantified using two indexes: The circularity index (CI) quantifies the roundness of organoids, and the intensity ratio (IR) is a measure of the presence of a central lumen. Non-CF organoids have a high CI and low IR compared to CF organoids. ROMA indexes perfectly discriminated 167 subjects with CF from 22 subjects without CF, making ROMA an appealing physiological CFTR assay to aid in CF diagnosis. Rectal biopsies can be routinely performed at all ages in most hospitals and tissue can be sent to a central lab for organoid culture and ROMA. In the future, ROMA might also be applied to test the efficacy of CFTR modulators in vitro. The aim of the present report is to fully explain the methods used for ROMA, to allow replication in other labs.

Rectal Organoid Morphology Analysis ROMA: A Diagnostic Assay in Cystic Fibrosis

This protocol describes rectal organoid morphology analysis (ROMA), a novel diagnostic assay for cystic fibrosis (CF). Morphological characteristics, namely the roundness (circularity index, CI) and the presence of a lumen (intensity ratio, IR), are a measure of CFTR function. Analysis of 189 subjects showed perfect discrimination between CF and non-CF.