作者没有确认。

1. 从血浆/血清中提取 miRNA
注意: 为了适当量化 miRNAs, 正确的 microRNA 隔离从血浆/血清是一个关键的步骤。要记住的关键一点是, 特别是由于存在不同的协议, 在处理示例时要遵循相同的工作流。在本议定书中, miRNA 从50µL 的血清中提取。不要使用超过200µL, 因为这限制了正确的提取过程。
<ol><li>准备血清或解冻冷冻样品。</li>
	<li>添加250µL (5 卷) 商业裂解试剂到50µL 血清。支持涡流的裂解。将管子与裂解液放在工作台的室温下5分钟。</li>
	<li>用3.5 µL 控制 (1.6 x 107拷贝/µL) 将裂解液钉在一起, 并用涡流混合。</li>
	<li>添加50µL 的氯仿 (即与起始血清样本相等的数量) 用于相分离。大力摇动管十五年代. 把管子放在工作台的室温下 2-3 分钟。</li>
	<li>为相分离, 离心机管在 1.2万 x g 在4°c 15 分钟。 注: 样品现在被分离成含 RNA 的上部水相, 白色相间, 和一个较低的, 粉红色的有机相。</li>
	<li>将含有 RNA 的上水相 (100 µL) 转移到新管中。不要转移任何白色相间的材料, 因为这伪造正确的 RNA 计数。</li>
	<li>添加150µL (即1.5x 的转移样品体积) 100% 乙醇。把这些材料吹打上下混合。不要将它们离心, 然后快速移动到下一步。</li>
	<li>将样品的250µL 在2毫升收集管的商用旋柱上。关上盖子。离心机柱在 > 8000 x g 在室温下十五年代. 丢弃流程。</li>
	<li>将700µL 的商用洗涤缓冲器编号1添加到自旋列。关闭盖子和离心机柱在 > 8000 x g 在室温下十五年代. 丢弃运行时的洗涤缓冲区。</li>
	<li>将500µL 的商用洗涤缓冲器编号2添加到自旋列。关闭盖子和离心机柱在 > 8, 000 x g 在室温下十五年代. 丢弃运行时的洗涤缓冲区。</li>
	<li>吸管500µL 80% 乙醇到自旋柱。关闭盖子, 离心机柱在 > 8, 000 x g 在室温下2分钟. 丢弃收集管与流经。</li>
	<li>将旋转柱转移到新的2毫升收集管。用打开的盖子将该柱离心在全速上晾干膜。将收集管丢弃, 并通过流经。</li>
	<li>将旋转柱转移到新的1.5 毫升收集管中。洗脱将30µL 的无 RNase 水直接应用于膜的中心。关上盖子。以全速离心柱1分钟。</li>
	<li>将 RNA 储存在-70 摄氏度。 注: 在-70 摄氏度至-20 摄氏度之间的 RNase 中纯化 rna 的储存确保 rna 的降解不超过1年。按照制造商的协议, 最低限度的 RNase 水稀释 RNA 是10µL. 使用少于10的µL 可能不足以使膜水化, 并降低总 RNA 的产量。</li>
</ol>2. 反向转录
注: 补充 DNA (cDNA) 是使用以下15µL 反向转录 (RT) 协议 (总反应量) 合成的。
<ol><li>在冰上解冻 RT 套件。尽可能长时间保持冷冻中的酶, 以防止它们在使用前降解和旋转。在冰上解冻 RT 底漆, 在使用前将其旋转。</li>
	<li>如表 1所述, 准备主组合。</li>
	<li>结合10µL 的主混合和5µL 提取 RNA 每井在96井板。</li>
	<li>离心机的井板在 2000 x g 在4°c 2 分钟。</li>
	<li>使用表 2中描述的热循环协议进行反向转录。 注: 如下所述, 可直接在数字 PCR 机中处理 cDNA (见材料表);或者, 它可以存储在摄氏-20 摄氏度。</li>
</ol>3. 液滴生成和数字 PCR
注: 使用以下40µL 协议进行了数字 PCR。
<ol><li>解冻的商业 dPCR 混合, 制备的 cDNA, 和 PCR 引物在冰上。在使用前不久将其离心。</li>
	<li>如表 3所述, 准备主组合。
</li>
	<li>添加 1.33 ul/井的 RT 产品和离心机简要。</li>
	<li>吸管 20 ul 样品入每井8井卡带 (中等列)。 注: 如果8井盒没有完全填充, 填补剩余的水井与非模板控制 (NTCs) 省略 cDNA (即RT 产品产生的水, 而不是提取 RNA) 或 dPCR 商业缓冲。NTC 作为污染控制。水不能在剩余的油井中使用, 因为这会损害液滴形成的质量。</li>
	<li>吸管 70 ul 的水滴产生油的探针进入油井 (底部的行) 的8井卡带。</li>
	<li>将垫片放在8井盒的顶部, 将卡带放在液滴发生器中。关闭液滴发生器。等待3指示灯呈稳定绿色。 注: 现在正在生成水滴。</li>
	<li>仔细和慢慢地转移 40 ul 的液滴形成的样品 (顶排) 成单独井的一个96井板由吹打。</li>
	<li>在样品上完成液滴形成后, 用 pierceable dPCR 箔在180摄氏度上密封 PCR 板4秒。</li>
	<li>将铝箔密封的 PCR 板放在循环仪中, 并按表 4所述循环, 以2.5 摄氏度/秒的坡道速率。</li>
</ol>4. 滴读和分析
<ol><li>将板从热循环仪转移到板架底座上。将板架顶部放在 pcr 盘上, 拧紧 pcr 盘。</li>
	<li>启动商业 dPCR 软件。</li>
	<li>在安装程序中输入示例名称、实验名称和目标。选择绝对量化。</li>
	<li>通过单击 "运行" 开始读取水滴。在使用水解探针时, 选择 "v" /"十六进制" 或 " VIC " 作为检测化学。 注意: 商业 dPCR 软件将自动分析数据。</li>
	<li>检查结果表中的水滴数以确保生成正确的雾滴。</li>
	<li>选择具有相同量化 miRNA 的所有井, 然后单击 "分析"。使用2D 印迹标记正滴, 并手动更正自动分析的数据 (图 1)。</li>
</ol>5. 对样品的正确计算
<ol><li>通过将样本乘以规范化因子 (NF) 来规范化样本。 NF = 中位 [线虫miR-39 测量]所有样品/[c. 线虫miR-39]给出样品
</li>
	<li>计算最终 miRNA 血清浓度, 调整稀释因子 (DF)。 miRNA最终= [miRNA]原始值x df x dfdPCR x dfEx 地方 miRNA原始价值= 商业 dPCR 软件量化的 miRNA; 在反向转录中模板的稀释因子; DFdPCR = 模板在 dPCR 中的稀释系数; DF = 稀释因子在萃取。</li>
</ol>6. 合成寡核苷酸稀释系列
<ol><li>解冻冻干合成寡核苷酸在冰上。简要地离心它。</li>
	<li>稀释核酸酶中的冻干合成寡核苷酸, 最终浓度为 10 pmol/µL. 简单地离心它。</li>
	<li>如表 5所述, 将其稀释在无核酸酶水中。离心稀释在每个稀释的步之间简要地在 8000 x g 十年代。</li>
	<li>按照步骤2中的说明继续进行反向转录, 并按照步骤3和步骤4中的说明对样本进行数字 PCR 分析。</li>
</ol>

<ol>
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作者没有什么可透露的。

数字 pcr 是一种相对新颖的 pcr 终点法, 它允许在样品中直接对核酸进行绝对量化。该方法具有特殊的优点, 包括可变性降低, 每日重现性增加, 灵敏度11,12。进一步, 由于样品分成大约2万个单一反应和端点分析, dPCR 比定量 rt-pcr16更健壮对干扰物质在 pcr。这些品质在 dPCR 使它成为一个有吸引力的替代定量 rt-pcr 作为诊断工具的量化。由于循环 miRNAs 经常存在于低血清浓度, 到目前为止, 科学家们已经被挑战, 以适当量化 miRNAs 的 PCR9。另一方面, dPCR 可以适当地量化甚至低 miRNA 表达的血清, 减轻的问题在低计数 miRNA 量化17。dPCR 的能力直接给予计数/µL, 即使在一个非常低的 miRNA 表达, 从而使它成为一个有吸引力的诊断工具, 心血管研究社区 miRNA 生物标志物研究。由于在计数/µL 中的浓度可以乘以稀释因子用于提取, RT 反应, 和 dPCR, 有可能达到确切数量的拷贝在1µL 的血清。这里提出的工作流可以在一个盘子上最多96个样本进行, 因此提供了一个大型心血管研究的工具。尽管 dPCR 在定量 rt-pcr 方面表现出一些优势, 但在心血管试验中 miRNAs 的定量化尚未得到常规的应用。此外, 还缺乏标准化的数据规范化程序。
在这种方法中, 我们证明, dPCR, 结合荧光水解探针, 使循环 miRNAs 的具体直接绝对量化与心血管疾病有关。通过这份报告, 我们的目标是通过dPCR 来证明一个优化的 miRNA 检测协议, 并确认 dPCR 在定量 rt-pcr 中的优越性。
我们确认了良好的线性 dPCR 展品和低限度的检测和量化的 dPCR 量化 miRNAs。通过合成寡核苷酸对样品进行峰值处理, 可以实现样品的规范化, 从而调整萃取效率和样品对样品的变异。
总之, 数字 PCR 技术水平和诊断电位均具有优越性, 是目前 miRNA 量化的最佳方法, 可进一步用于大型多中心心血管 miRNA 生物标志物的研究。

循环 miRNAs 已被确定为许多疾病, 包括心血管疾病1的有前景的标志。miRNAs 是小的, 非编码单链 RNA 分子 (大约22核苷酸长)通过信使 RNA 翻译的改变和影响基因表达2参与后转录调控,并被释放到循环中的生理和病理状态。具体 miRNAs 的定量表达可以与病理学相联系, 一些 miRNAs 表现出较高的组织和疾病特异性1。在心血管疾病中, miRNAs 已成为具有吸引力的候选生物标志物, 因为它们在血清中明显稳定, 可以在 PCR 方法3的帮助下很容易量化。miRNAs 作为心肌梗死的生物标志物的潜在价值在小的研究中得到了评价, 但在大群群中的验证却缺乏2。例如, miR-499 被发现在心肌肌肉中表现得很高, 在 AMI4、5、6中表现出明显的增加。此外, 它调节程序性细胞死亡 (凋亡) 和心肌细胞的分化, 因而参与了 AMI7后的几个机制。除了一些小的研究报告 miRNAs 的优势和增量价值的诊断 AMI, 高灵敏度的心脏肌钙蛋白的优越性或平等尚未证明在大规模研究2,5 ,6,8。因此, 需要对大型群体进行更多前瞻性研究, 以评估 miRNAs 的潜在诊断价值。此外, miRNA 量化的方法需要优化和标准化使用可比较的协议9。标准化化验可以减少不一致的结果, 并可能有助于 miRNAs 成为潜在的生物标志物的常规临床应用, 因为生物标志物需要量化的可重复的方式, 以确保其临床适用性。
最近, dPCR 作为一个终点分析被引入。它划分样品入大约2万个各自的反应10。dPCR 系统然后利用数学泊松统计分析的荧光信号 (正负反应), 使绝对量化没有标准曲线10。将 dPCR 与荧光水解探针相结合, 使 miRNAs 的高度特异直接绝对量化成为可能。数字聚合酶链反应显示出优越的技术品质 (包括可变性下降, 日到日重现, 高度线性度和高灵敏度), 以量化 miRNA 水平在循环比定量实时 PCR10,11。这些优越的技术品质可能有助于减轻目前使用循环 miRNAs 作为生物标志物的限制, 并可能导致在大型多中心心血管临床试验中建立 miRNAs 作为生物标记, 并作为一种诊断方法在一般。在先前的一项研究中, 我们最近应用 dPCR 对 AMI 患者循环 miRNAs 的绝对量化, 并能显示出优于 qPCR12定量 miRNAs 的诊断潜力。
在本刊物中, 我们想证明使用 dPCR 是一种准确和可重复的方法直接量化循环心血管 miRNAs。用数字 PCR 技术对血清中 miRNA 水平的绝对量化, 显示了在大型多中心心血管临床试验中的应用潜力。在本论文中, 我们详细描述了如何有效地执行数字 PCR 以及如何检测血清中的绝对 miRNA 拷贝数。

<table>
	<tbody>
		<tr>
			<td>RNA-Extraction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy Serum/Plasma Kit (50)</td>
			<td>Qiagen-Sample &#38; Assay Technologies, Hilden, Deutschland</td>
			<td>217184</td>
			<td>Kit for microRNA extraction. Kit contains commercial buffer RWT (called number one in the manuscript)&#160; and RPE (called number two in manuscript).</td>
		</tr>
		<tr>
			<td>miRNeasy Serum/Plasma Spike-in-Control; Syn-cel-miR-39 miRNA; 10pmol</td>
			<td>Qiagen-Sample &#38; Assay Technologies, Hilden, Deutschland</td>
			<td>219610</td>
			<td>Spike-in for normalisation , Sequence: 5&#39;-UCACCGGGUGUAAAUCAGCUUG-3&#39;</td>
		</tr>
		<tr>
			<td>Reverse Transcription</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Reverse Transcription Kit (1000 Reactions)</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4366597</td>
			<td>Kit for microRNA reverse transcription</td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Assays M</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4440887</td>
			<td>Assays used in reverse transcription</td>
		</tr>
		<tr>
			<td>hsa-miR-499 (750 RT/750 PCR rxns)</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4440887</td>
			<td>Assay Number 001352</td>
		</tr>
		<tr>
			<td>cel-miR-39 (750 RT/750 PCR rxns)</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4440887</td>
			<td>Assay Number 000200</td>
		</tr>
		<tr>
			<td>PCR Plate, 96-well, segmented, semi-skirted</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>AB0900</td>
			<td>96 well plate for reverse transcription</td>
		</tr>
		<tr>
			<td>Microseal &#8216;B&#8217; seal Seals</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>MSB1001</td>
			<td>Foil to ensure proper storage</td>
		</tr>
		<tr>
			<td>C1000 Touch Thermal Cycler</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1851196</td>
			<td>Cycler used for reverse transcription</td>
		</tr>
		<tr>
			<td>Droplet Digital PCR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 nmole RNA oligo hsa-miR-499-5p</td>
			<td>Integrated DNA Technologies</td>
			<td>Custom</td>
			<td>Sequence: 5&#39;-phos-UUAAGACUUGCAGUGAUGUUU-3&#39;</td>
		</tr>
		<tr>
			<td>ddPCR Supermix for Probes (No dUTP)</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863024</td>
			<td>Supermix used in droplet generation</td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Assays</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4440887</td>
			<td>Assays used in digital PCR (fluorescent hydrolysis probe)</td>
		</tr>
		<tr>
			<td>hsa-miR-499 (750 RT/750 PCR rxns)</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4440887</td>
			<td>Assay Number 001352, commercial primers</td>
		</tr>
		<tr>
			<td>cel-miR-39 (750 RT/750 PCR rxns)</td>
			<td>Applied Biosystems, Inc., Foster City, CA, USA</td>
			<td>4440887</td>
			<td>Assay Number 000200, commercial primers</td>
		</tr>
		<tr>
			<td>DG8 Cartridges and Gaskets</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1864007</td>
			<td>Cartridge takes up to 8 samples for droplet generation</td>
		</tr>
		<tr>
			<td>DG8 Cartridge Holder</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863051</td>
			<td>Holds cartridges in droplet generation</td>
		</tr>
		<tr>
			<td>Droplet Generation Oil for Probes</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863005</td>
			<td>Oil used in droplet generation</td>
		</tr>
		<tr>
			<td>ddPCR 96-Well Plates</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>12001925</td>
			<td>96 well plate for ddPCR</td>
		</tr>
		<tr>
			<td>PCR Plate Heat Seal, foil, pierceable</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1814040</td>
			<td>Pierceable foil, compatible with droplet reader</td>
		</tr>
		<tr>
			<td>ddPCR Droplet Reader Oil</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863004</td>
			<td>Oil used in droplet reading</td>
		</tr>
		<tr>
			<td>QX100 or QX200 Droplet Generator</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863002</td>
			<td>Droplet Generator, generates the droplets from sample/oil emulsion</td>
		</tr>
		<tr>
			<td>PX1 PCR Plate Sealer</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1814000</td>
			<td>Seals the plate before PCR</td>
		</tr>
		<tr>
			<td>C1000 Touch Thermal Cycler</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1851196</td>
			<td>Cycler used for ddPCR</td>
		</tr>
		<tr>
			<td>QX100 or QX200 Droplet Reader</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863003</td>
			<td>Reads PCR-positive and PCR-negative droplets with an optical detector</td>
		</tr>
		<tr>
			<td>ddPCR Buffer Control for Probes</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1863052</td>
			<td>Blank control&#160;and to fill up the remaining wells of 8-well cassette</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QuantaSof Software</td>
			<td>Bio-Rad Laboratories, Inc., Hercules, CA, USA</td>
			<td>1864011</td>
			<td>Program for droplet reading</td>
		</tr>
		<tr>
			<td>Prism Windows 5</td>
			<td>GraphPad Software Inc., La Jolla, CA, USA</td>
			<td></td>
			<td>Program for statistical analysis</td>
		</tr>
	</tbody>
</table>

digital pcr for quantifying circulating micrornas in acute myocardial infarction and cardiovascular disease

循环血清 microRNAs (miRNAs) 表明, 作为心血管疾病和急性心肌梗死 (AMI) 的标志物, 从心血管细胞释放到循环。循环 miRNAs 高度稳定, 可量化。具体 miRNAs 的定量表达可以与病理学相联系, 一些 miRNAs 表现出较高的组织和疾病特异性。为心血管疾病寻找新的生物标志物对医学研究具有重要意义。最近, 数字聚合酶链反应 (dPCR) 已经发明。dPCR, 结合荧光水解探针, 使特定的直接绝对量化。与定量聚合酶链反应 (qPCR) 相比, dPCR 具有优异的技术质量, 包括低变异性、高线性度和高灵敏度。因此, dPCR 是一种更准确和可重复的方法直接量化 miRNAs, 特别是用于大型多中心心血管临床试验。在本论文中, 我们描述了如何有效地执行数字 PCR, 以评估血清样本中的绝对拷贝数。

数字 PCR 结合荧光水解探针使研究人员可以直接量化的绝对数量的特定 miRNAs 在拷贝/µL。当样品在 dPCR 被划分在大约2万个单独 PCR 反应, dPCR 不需要技术复制10。dPCR 系统利用数学泊松统计分析的荧光信号 (不同的正面和负面的反应), 使绝对量化, 而无需标准曲线10。为了正确地计算浓度, 需要一个可接受的水滴计数 (> 1.5万)。没有模板控件确保排除相当多的非特异性放大。在分析中所包含的所有样本均采用相同的体积、方法和 PCR 协议进行处理, 以增强所得结果的有效性。在 dPCR 中获得的浓度是通过对所有样本的中值规范化过程进行规范化的, 这些样品用合成的尖 miR-3913进行分析。将数据规范化到被测量的 miR-39 的量中 , 可以纠正 RNA 提取效率中样本对样本的变异, 并作为 PCR 反应控制, 进一步增加结果的有效性。血清 miRNAs 规范化尚无确定的金标准;然而, 外源控制, 如 miR-39线虫是规范化程序的优雅解决方案, 因为内源性 miRNAs 经常改变在各种疾病状态9。
分析血清标本获得经皮冠状动脉介入治疗 (pci), 8 小时后 pci, 16 小时 pci 后, 从患者急性 ST 段抬高心肌梗死 (STEMI)。STEMI 患者表现出严重的缺血, 因此有资格评估新的缺血生物标志物。为评价 miR-499 释放的动力学和 miR-499 作为心血管病缺血生物标志物的潜在应用, miR-499 对三个不同时间点14的患者进行了 dPCR 分析。
图 1展示了通过软件计算最终 miRNA 浓度的正水滴的选择。样本中阳性的分数决定了商业 dPCR 软件所计算的拷贝/µL 的浓度。结果也可以在2D 图中进行可视化, 并且可以用圆圈手动标记阳性。浓度只有在 NTCs 的非特异性放大时才会被考虑。
图 2显示了合成 miRNA 寡核苷酸 has-miR-499-5p 稀释系列中重复的线性度和拟合性。8步稀释系列是从2500个拷贝/µL 到0个拷贝/µL 进行的。计算出的预期拷贝/µL 如表 5所述, 假定为 100% RT 和数字 PCR 效率。在这个设置中, 数字 PCR 显示了高度的线性度 (r2 = 0.99)。在图 2中还显示了检测的极限 (LoD = 0.12) 和量化 (loq 矿用 = 0.23) 的限制, 表明即使是低 miRNA 表达式也能成功检测到。因此, dPCR 可以用来量化甚至低血清水平的循环 miRNAs。根据 Forootan等方法, 计算了检测的极限 (LoD) 和量化 (loq 矿用) 的极限。15并且被定义为 LoD = LoB + 1.645 x σ低浓度样品, 其中空白 (LoB) 的极限被计算作为 LoB = 平均空白+ 1.645 x σ空白。loq 矿用然后估计运行复制标准曲线15。为了确保 miRNAs 的可重现量化, 只有在 LoD 和 loq 矿用以上的 miRNA 浓度才被视为有效的精确值。
图 3显示了 ST 升高心肌梗死 (STEMI) 患者 miR-499 水平的代表性结果, 每一次点n = 16。样本在经皮介入 (pci) (t = 0), 8 小时后 pci (t = 8) 和 16 h 后 pci (t = 16), 和 miR-499 水平比较的 miR-499 水平的患者稳定冠心病 (CAD, n= 20)。患者的主要特点可以在表 6中找到。在心肌梗死后的第一个8小时内, miR-499 进入血清后, miR-499 水平在16小时后再次下降。在 STEMI (t = 0) 开始时, 可能已经看到了增加的趋势, 与 CAD 患者相比, miR-499 水平的显著增加在 STEMI (t = 8, p < 0.01) 比较 miR-499 水平时可以看到8小时。miR-499 水平的稳定 CAD 患者。接收机操作曲线 (ROC) 显示 miR-499 作为一种新的生物标志物的可能效用, 用于区分 CAD 患者和 STEMI 患者 [曲线下的区域 (联合自卫队) = 0.62 在经皮介入治疗 (PCI) 前进行的取样, 联合自卫组 =0.75 对于在 pci 后服用8小时的样品和联合自卫组 = 0.78, 用于在 pci 后服用16小时的样本与 CAD 患者的血清 miR-499 水平相比。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57950/57950fig1.jpg"> 图 1: 在重复执行的稀释系列中选择正水滴.(A) 阈值是在负水滴上方手动设置的。(B) 结果在2D 印迹中被可视化, 通过盘旋选择阳性。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57950/57950fig1large.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/57950/57950fig2.jpg"> 图 2: 合成 miR-499 寡核苷酸稀释系列.(A) 测量结果是重复进行的。这些数据被显示为每µL 的绝对拷贝. 线性回归和拟合 (r2值) 的优点。这些数据以 "平均" 扫描电镜显示. (B) 本小组展示了检测的限度 (LoD) 和量化的限度 (loq 矿用)。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57950/57950fig2large.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/57950/57950fig3.jpg"> 图 3: 与稳定型冠状动脉患者相比, 经皮冠状动脉介入治疗 (pci)、pci 后8小时、pci 后16小时的 ST 段抬高心肌梗死 (n = 16) 患者循环血清 miR-499-5p 的量化疾病 (n = 20).(A) 循环血清 miR-499-5p 表示为平均值, 其标准误差与以单向方差分析法计算的相应p值, 其次为 Bonferroni后试验 (p < 0.05 被认为是统计学意义)。(B) 对a组的数据执行接收器操作特性曲线 (ROC) 分析。中华民国的分析表明, 生物标志物的敏感性和特异性。此外, 还显示了曲线 (联合自卫队) 值下的相应区域。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57950/57950fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="4">样本量</td>
			<td></td>
		</tr>
<tr>
<td colspan="4">n = 1</td>
			<td>/以及 [ul]</td>
		</tr>
<tr>
<td colspan="4">核酸酶水</td>
			<td>4.16</td>
		</tr>
<tr>
<td colspan="4">10x 反向转录缓冲器</td>
			<td>1。5</td>
		</tr>
<tr>
<td colspan="4">100nM dNTP</td>
			<td>0.15</td>
		</tr>
<tr>
<td colspan="4">RNAse 抑制剂</td>
			<td>0.19</td>
		</tr>
<tr>
<td colspan="4">特定 RT 底漆</td>
			<td>3</td>
		</tr>
<tr>
<td colspan="4">
商业逆转录酶50 U/ul</td>
			<td>1</td>
		</tr>
<tr>
<td colspan="4">主混合总计</td>
			<td>10</td>
		</tr>
</tbody></table>表 1: 反转转录试剂组合。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>温度</td>
			<td>时间</td>
		</tr>
<tr>
<td>16°c</td>
			<td>30分钟</td>
		</tr>
<tr>
<td>42°c</td>
			<td>30分钟</td>
		</tr>
<tr>
<td>85°c</td>
			<td>5分钟</td>
		</tr>
<tr>
<td>4°c</td>
			<td>∞</td>
		</tr>
</tbody></table>表 2: 反向转录的热循环条件。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="4">样本量</td>
			<td></td>
		</tr>
<tr>
<td colspan="4">n = 1</td>
			<td>/以及 [ul]</td>
		</tr>
<tr>
<td colspan="4">核酸酶水</td>
			<td>7.67</td>
		</tr>
<tr>
<td colspan="4">商业 dPCR 混合</td>
			<td>10</td>
		</tr>
<tr>
<td colspan="4">20x 特殊水解底漆/探头</td>
			<td>1</td>
		</tr>
<tr>
<td colspan="4">主混合总计</td>
			<td>18.67</td>
		</tr>
</tbody></table>表 3: 数字 PCR 试剂组合。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>温度</td>
			<td>时间</td>
		</tr>
<tr>
<td>95°c</td>
			<td>10分钟</td>
		</tr>
<tr>
<td>94°c</td>
			<td>30秒 (x40)</td>
		</tr>
<tr>
<td>60°c</td>
			<td>1分钟</td>
		</tr>
<tr>
<td>98°c</td>
			<td>10分钟</td>
		</tr>
<tr>
<td>4°c</td>
			<td>∞</td>
		</tr>
</tbody></table>表 4: 数字 PCR 的热循环条件。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>管</td>
			<td>转移合成寡核苷酸 miR-499 (µL)</td>
			<td>稀释剂 (µL)</td>
			<td>miRNA (复印件/µL)</td>
			<td>预期的拷贝/µL 在 RT 中</td>
			<td>dPCR 中的预期副本/µL</td>
		</tr>
<tr>
<td>源语言</td>
			<td></td>
			<td></td>
			<td>6.022 x 1012
</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>1</td>
			<td>10</td>
			<td>990</td>
			<td>6.022 x 1010
</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>2</td>
			<td>10</td>
			<td>990</td>
			<td>6.022 x 108
</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>3</td>
			<td>10</td>
			<td>990</td>
			<td>6.022 x 106
</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>4</td>
			<td>18。7</td>
			<td>981。3</td>
			<td>1.128 x 105
</td>
			<td>37600</td>
			<td>2500</td>
		</tr>
<tr>
<td>5</td>
			<td>40</td>
			<td>60</td>
			<td>4.511 x 104
</td>
			<td>15040</td>
			<td>1000</td>
		</tr>
<tr>
<td>6</td>
			<td>50</td>
			<td>50</td>
			<td>2.256 x 104
</td>
			<td>7520</td>
			<td>500</td>
		</tr>
<tr>
<td>7</td>
			<td>40</td>
			<td>160</td>
			<td>4.511 x 103
</td>
			<td>1504</td>
			<td>100</td>
		</tr>
<tr>
<td>8</td>
			<td>50</td>
			<td>50</td>
			<td>2.256 x 103
</td>
			<td>752</td>
			<td>50</td>
		</tr>
<tr>
<td>9</td>
			<td>40</td>
			<td>160</td>
			<td>4.511 x 102
</td>
			<td>150。4</td>
			<td>10</td>
		</tr>
<tr>
<td>10</td>
			<td>50</td>
			<td>150</td>
			<td>1.128 x 102
</td>
			<td>37。6</td>
			<td>2。5</td>
		</tr>
<tr>
<td>11</td>
			<td>0</td>
			<td>50</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr>
</tbody></table>表 5: miR-499 的合成稀释系列。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>特征</td>
			<td>所有</td>
			<td>稳定 CAD 患者 (n=20)</td>
			<td>STEMI 患者 (n = 24)</td>
			<td>p 值</td>
		</tr>
<tr>
<td>年龄</td>
			<td>64.7 @ 11。9</td>
			<td>66.7 @ 13。1</td>
			<td>62.2 @ 9。9</td>
			<td>0.2810</td>
		</tr>
<tr>
<td>男性</td>
			<td>77.8%</td>
			<td>65%</td>
			<td>93.8%</td>
			<td>0.0392</td>
		</tr>
<tr>
<td>Dm</td>
			<td>33.3%</td>
			<td>40%</td>
			<td>25%</td>
			<td>0.3428</td>
		</tr>
<tr>
<td>HTN</td>
			<td>61.1%</td>
			<td>60%</td>
			<td>62.5%</td>
			<td>0.8785</td>
		</tr>
<tr>
<td>血脂 异常</td>
			<td>38.9%</td>
			<td>65%</td>
			<td>6.3%</td>
			<td>0.0003</td>
		</tr>
<tr>
<td>吸烟者</td>
			<td>47.2%</td>
			<td>55%</td>
			<td>37.5%</td>
			<td>0.3796</td>
		</tr>
<tr>
<td>家庭历史</td>
			<td>25%</td>
			<td>35%</td>
			<td>12.5%</td>
			<td>0.1213</td>
		</tr>
<tr>
<td>超重</td>
			<td>58.3%</td>
			<td>55%</td>
			<td>62.5%</td>
			<td>0.6501</td>
		</tr>
<tr>
<td>血清肌酐 (毫克/dL)</td>
			<td>0.94 (0.83-1)</td>
			<td>0.98 (0.82-1.01)</td>
			<td>0.93 (0.83-1)</td>
			<td>0.7255</td>
		</tr>
<tr>
<td>CK 峰值 (U/I)</td>
			<td>161 (102-611)</td>
			<td>126 (81-161)</td>
			<td>492 (228-3208)</td>
			<td>0.0004</td>
		</tr>
<tr>
<td>cTnT 峰 (吴/升)</td>
			<td>322.5 (23.8-4533)</td>
			<td>18 (7-25.3)</td>
			<td>2492 (240-5586)</td>
			<td>0.0002</td>
		</tr>
<tr>
<td>LVEF</td>
			<td>45% (40%-50%)</td>
			<td>45% (32.5%-55%)</td>
			<td>45% (45%-50%)</td>
			<td>0.9596</td>
		</tr>
<tr>
<td colspan="4">值被显示为平均值。中值 (第二十五至第七十五百分比范围) 或%</td>
			<td></td>
		</tr>
<tr>
<td>DM: 糖尿病</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>HTN: 高血压</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>肌酸激酶</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>cTnT: 心肌肌钙蛋白 T</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td colspan="2">LVEF: 左心室射血分数</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
</tbody></table>表 6: 主要病人特点。

Watch this Scientific Journal Video about Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease at JoVE.com

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

循环 microRNAs 表明, 作为心血管疾病和急性心肌梗死的标志物的承诺。在本研究中, 我们描述了一个 miRNA 提取, 反向转录和数字 PCR 的协议, 以绝对量化的 miRNAs 在心血管疾病患者的血清。

数字 PCR 在急性心肌梗死和心血管疾病中量化循环 MicroRNAs 的临床观察

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...

digital-pcr-for-quantifying-circulating-micrornas-acute-myocardial

Nanyang Technological University

Research

JoVE Journal

Medicine

8.0K 次观看.  University of Freiburg. 循环 microRNAs 表明, 作为心血管疾病和急性心肌梗死的标志物的承诺。在本研究中, 我们描述了一个 miRNA 提取, 反向转录和数字 PCR 的协议, 以绝对量化的 miRNAs 在心血管疾病患者的血清。

视频: Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

大鼠急性心肌梗死

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

科研

医学

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

MRI和PET心肌梗死小鼠模型

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

心肌梗死和功能预后评估的猪

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

在诱导小鼠心肌梗死的微创手术过程

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

左冠状动脉前降支的小鼠结扎:对心肌梗死后重塑和心衰模型

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

急性心肌梗死的猪模型主要成果评估

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

小鼠急性心肌梗死的实时压力容积分析

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

A Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

研究小鼠心肌梗死的低温损伤模型

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

诱导大鼠心肌梗死的半微创方法及独立工作心脏系统对心脏功能的评估

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

Murine 模型心肌梗塞后 MSC 加载注射水凝胶的心内移植

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...