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 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>simvastatin (pro-drug)</td><td>Cayman Chemicals</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>lovastatin (pro-drug)</td><td>Cayman Chemicals</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>rosuvastatin</td><td>Cayman Chemicals</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>atorvastatin</td><td>Cayman Chemicals</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>fluvastatin</td><td>Cayman Chemicals</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>ezetimibe (pro-drug)</td><td>Cayman Chemicals</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>Niacin</td><td>MilliporeSigma</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>fibrate</td><td>MilliporeSigma</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;C</td></tr><tr><td>omega-3 fatty acid</td><td>MilliporeSigma</td><td></td><td>store 100 &amp;micro;l aliquots at -20 &amp;deg;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 measurement</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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14.6K 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

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high&#8208;resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high&#8208;throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

More than half of proteins are small proteins (molecular mass &#60;200 kDa) that are challenging for both electron microscope imaging and three-dimensional reconstructions. Optimized negative staining is a robust and high-throughput protocol to obtain high contrast and relatively high resolution (~1 nm) images of small asymmetric proteins or complexes under different physiological conditions.

优化负染：通过电子显微镜检查小和不对称蛋白结构的高通量协议

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of ...

科研

环境科学

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 ...

医学

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

生物化学

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of methods. Here, we describe two important approaches used for this purpose. First, we describe how to enrich tissues and cells with cholesterol using cyclodextrin saturated with cholesterol using cerebral arteries (tissues) and hippocampal neurons (cells) as examples. This approach can be used for any type of tissue, cells, or cell lines. An alternative approach for cholesterol enrichment involves the use of low-density lipoprotein (LDL). The advantage of this approach is that it uses part of the natural cholesterol homeostasis machinery of the cell. However, whereas the cyclodextrin approach can be applied to enrich any cell type of interest with cholesterol, the LDL approach is limited to cells that express LDL receptors (e.g., liver cells, bone marrow-derived cells such as blood leukocytes and tissue macrophages), and the level of enrichment depends on the concentration and the mobility of the LDL receptor. Furthermore, LDL particles include other lipids, so cholesterol delivery is nonspecific. Second, we describe how to enrich Xenopus oocytes with cholesterol using a phospholipid-based dispersion (i.e., liposomes) that includes cholesterol. Xenopus oocytes constitute a popular heterologous expression system used for studying cell and protein function. For both the cyclodextrin-based cholesterol enrichment approach of mammalian tissue (cerebral arteries) and for the phospholipid-based cholesterol enrichment approach of Xenopus oocytes, we demonstrate that cholesterol levels reach a maximum following 5 min of incubation. This level of cholesterol remains constant during extended periods of incubation (e.g., 60 min). Together, these data provide the basis for optimized temporal conditions for cholesterol enrichment of tissues, cells, and Xenopus oocytes for functional studies aimed at interrogating the impact of cholesterol enrichment.

Enrichment of Mammalian Tissues and Xenopus Oocytes with Cholesterol

Two methods of cholesterol enrichment are presented: the application of cyclodextrin saturated with cholesterol to enrich mammalian tissues and cells, and the use of cholesterol-enriched phospholipid-based dispersions (liposomes) to enrich Xenopus oocytes. These methods are instrumental for determining the impact of elevated cholesterol levels in molecular, cellular, and organ function.

哺乳动物组织和异种卵母细胞与胆固醇的丰富

Cholesterol enrichment of mammalian tissues and cells, including Xenopus oocytes used for studying cell function, can be accomplished using a variety of ...

Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and atherosclerosis. Although there are several methods for analyzing plasma lipoproteins, ultracentrifugation is still one of the most popular and reliable methods. Because of its intact separation procedure, the lipoprotein fractions isolated by this method can be used for analysis of lipoproteins, apolipoproteins, proteomes, and functional study of lipoproteins with cultured cells in vitro. Here, we provide a detailed protocol to isolate seven lipoprotein fractions including VLDL (d&#60;1.006 g/mL), IDL (d=1.02 g/mL), LDLs (d=1.04 and 1.06 g/mL), HDLs (d=1.08, 1.10, and 1.21 g/mL) from rabbit plasma using sequential floating ultracentrifugation. In addition, we introduce the readers how to analyze apolipoproteins such as apoA-I, apoB, and apoE by SDS-PAGE and Western blotting and show representative results of lipoprotein and apolipoprotein profiles using hyperlipidemic rabbit models. This method can become a standard protocol for both clinicians and basic scientists to analyze lipoprotein functions.

Several methods have been used for analyzing plasma lipoproteins; however, ultracentrifugation is still one of the most popular and reliable methods. Here, we describe a method regarding how to isolate lipoproteins from plasma using sequential density ultracentrifugation and how to analyze the apolipoproteins for both diagnostic and research purposes.

超中心对等离子脂蛋白的分离和分析

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and ...

Formulation and Characterization of Bioactive Agent Containing Nanodisks

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle&#39;s lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk&#160;particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin&#160;B as the hydrophobic bioactive agent.

Here, we describe the production and characterization of bioactive agents containing nanodisks. Amphotericin B nanodisks are taken as an example to describe the protocol in a stepwise manner.

含纳米片生物活性剂的配制与表征

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...

使用 3D 荧光脂滴重建评估肝脂肪变性

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing dysfunction caused by free fatty acids (FAs). The liver, given its critical role in the body's fat metabolism, is persistently threatened by the intracellular accumulation of LDs in the form of both microvesicular and macrovesicular hepatic steatosis. The histologic characterization of LDs is typically based on lipid-soluble diazo dyes, such as Oil Red O (ORO) staining, but a number of disadvantages consistently hamper the use of this analysis with liver specimens. More recently, lipophilic fluorophores 493/503 have become popular for visualizing and locating LDs due to their rapid uptake and accumulation into the neutral lipid droplet core. Even though most applications are well-described in cell cultures, there is less evidence demonstrating the reliable use of lipophilic fluorophore probes as an LD imaging tool in tissue samples. Herein, we propose an optimized boron dipyrromethene (BODIPY) 493/503-based protocol for the evaluation of LDs in liver specimens from an animal model of high-fat diet (HFD)-induced hepatic steatosis. This protocol covers liver sample preparation, tissue sectioning, BODIPY 493/503 staining, image acquisition, and data analysis. We demonstrate an increased number, intensity, area ratio, and diameter of hepatic LDs upon HFD feeding. Using orthogonal projections and 3D reconstructions, it was possible to observe the full content of neutral lipids in the LD core, which appeared as nearly spherical droplets. Moreover, with the fluorophore BODIPY 493/503, we were able to distinguish microvesicles (1 &#181;m &lt; d &#8804; 3 &#181;m), intermediate vesicles (3 &#181;m &lt; d &#8804; 9 &#181;m), and macrovesicles (d &gt; 9 &#181;m), allowing the successful discrimination of microvesicular and macrovesicular steatosis. Overall, this BODIPY 493/503 fluorescence-based protocol is a reliable and simple tool for hepatic LD characterization and may represent a complementary approach to the classical histological protocols.

作者聚焦： 使用 3D 重建分析荧光染色脂滴用于肝脂肪变性评估  

Herein, we demonstrate an optimized BODIPY 493/503 fluorescence-based protocol for lipid droplet characterization in liver tissue. Through the use of orthogonal projections and 3D reconstructions, the fluorophore allows for successful discrimination between microvesicular and macrovesicular steatosis and may represent a complementary approach to the classical histological protocols for hepatic steatosis assessment.

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing ...

Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

We describe here a fluorometric cell-free biochemical assay for determination of HDL-lipid peroxidation. This rapid and reproducible assay can be used to determine HDL function in large scale studies and can contribute to our understanding of HDL function in human disease.

无细胞生化荧光酶法测定高密度脂蛋白脂质过氧化的高通量测定

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular ...

降血脂药物对胆固醇颗粒形态的差异化作用

摘要

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优化负染：通过电子显微镜检查小和不对称蛋白结构的高通量协议

胆固醇外流检测

微 Rna 对原生脂蛋白颗粒的富集及其与绝对相对微 Rna 含量及细胞转移速率的测定

哺乳动物组织和异种卵母细胞与胆固醇的丰富

超中心对等离子脂蛋白的分离和分析

含纳米片生物活性剂的配制与表征

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

荧光染色脂滴分析与3D重建，用于肝脂肪变性评估

无细胞生化荧光酶法测定高密度脂蛋白脂质过氧化的高通量测定