这项工作得到国家杰出青年学者基金会 (81625002)、中国国家自然科学基金 (81470389、81270282、81601238) 的支持, 上海市学术研究领导 (18XD1402400) 项目教育统筹委员会高峰临床医学资助 (20152209), 上海中药肾康医院发展中心 (16CR3034A), 上海交通大学 (YG2013MS42), 上海交通大学医学院 (15ZH1003 和 14XJ10019),上海航海项目 (18YF1413000) 和蚌埠医学院研究生创新项目 (Byycx1722)。我们感谢乌尔博士以前在我们实验室的帮助。

所有动物实验都是根据国家卫生研究院关于使用实验动物的指导方针进行的, 并得到了研究所动物伦理委员会的批准。所有的实验都使用雄性 C57BL/6J 小鼠 (8-10 周龄)。小鼠被安置了在无病原体条件下24°c 4 °c, 在 12 h 光/黑暗周期, 与自由获得水和食物。
1. 腺病毒溶液的制备
<ol><li>纯化腺病毒溶液到达后, 将溶液贮存在-80 摄氏度的冷冻机中。 注: 腺病毒注射液是在存活的小鼠体内进行的。为确保腺病毒载体的不育, 制备并纯化了9、10的腺病毒 (荧光素酶、vdr 或 shRNA 靶向 vdr)。</li>
	<li>在手术当天, 从-80 °c 冰箱取出纯化的腺病毒溶液 (3 x 1010斑块形成单元 [pfu]/毫升), 并在冰上解冻腺病毒载体溶液。</li>
	<li>戴上面罩、消毒手套和无菌长袍。</li>
	<li>准备一支50µL 的哈密尔顿注射器, 在手术前一天就被消毒了。 注: 为杀菌, 哈密尔顿注射器包裹在纱布和放置在高温/高压灭菌器。选择 "固体灭菌" 模式, 按 "启动" 按钮开始灭菌。</li>
	<li>纯化腺病毒溶液完全解冻后, 用75% 乙醇喷洒含有腺病毒溶液的管, 将腺病毒溶液移入层流无菌罩中。</li>
	<li>在层流无菌罩, 吸入50µL 腺病毒溶液使用哈密尔顿注射器没有针, 其次是附着30克针的汉密尔顿注射器。</li>
	<li>按住注射器与30克针向上, 并轻轻推注射器的柱塞, 直到针充满腺病毒溶液 (确认由第一溶液的外观下降从针尖)。 注: 用大约45µL 溶液填充30克针, 所以现在几乎没有解决方案是可见的注射器。</li>
	<li>使用上述准备好的注射器, 仔细吸入另30µL 腺病毒溶液, 并将松散的上限注射器放在冰上供以后使用。</li>
</ol>2. 麻醉和手术准备
<ol><li>在异氟醚蒸发器中加入20毫升异氟醚, 并将异氟醚蒸发器与氧气罐连接。</li>
	<li>打开阀门, 让氧气从氧气罐流向异氟醚蒸发器。通过在异氟醚蒸发器中的氧气流量监测器, 保持2升/分的氧气流量。</li>
	<li>用4% 异氟醚在100% 氧 (2 升/分) 中诱导麻醉, 在与异氟醚蒸发器相连的塑料笼中2分钟。</li>
	<li>从塑料笼中取出麻醉鼠标, 并将其固定在层流消毒罩中俯卧位置的塑料平台上, 并通过鼻锥在100% 氧 (2 升/分) 内保持麻醉, 2% 异氟醚。</li>
	<li>确认足够的麻醉, 没有脚趾捏反应。</li>
	<li>将无菌眼霜应用于每只眼睛, 以保护角膜不受干燥。</li>
	<li>剃掉胸部和上腹部。将商业上可用的脱毛膏涂抹在剃须的地方1分钟. 去除脱毛霜和剩余的毛皮与湿纱布。</li>
	<li>用3的碘洗必泰消毒 (如 entoiodine) 消毒手术部位 (左下半部分)。用不育的褶皱覆盖手术部位。</li>
</ol>3. 心肌注射腺病毒在小鼠心脏中的研究
<ol><li>脱下手套, 戴上一双新的无菌手套。</li>
	<li>消毒钳, 微型蚊子止血, 一对手术剪刀, 和一个针持有人在运行前的高温/高压灭菌器 (见步骤1.4 注意)。</li>
	<li>沿着连接剑突和腋窝的线做一个0.5 厘米的皮肤切口。用镊子和微蚊止血直接解剖胸主和胸肌小肌肉。</li>
	<li>通过缩回胸主和胸小肌肉来暴露肋间空间。刺穿并打开第四肋间空间 (或观察最宽的肋骨空间) 与微蚊子止血。</li>
	<li>将心脏推向切口, 用非显性手的食指轻轻按压胸壁右侧, 将心脏外侧。</li>
	<li>用非显性手的食指和拇指轻轻地保护外在的心脏。</li>
	<li>通过用占主导地位的腺病毒 (图 1C) 填充的哈密尔顿注射器, 将30µL 腺病毒溶液注入三个部位 (腹侧、背部和左心室侧壁) 的左心室心肌。</li>
	<li>注射完成后, 立即将心脏放回胸腔内。</li>
	<li>通过轻轻按压胸壁向皮肤切口部位, 手动疏散胸腔内的空气。 注: 成功的空气疏散可以通过拍打的胸部少校和胸小肌肉造成的驱逐空气证明。</li>
	<li>用5-0 丝缝线缝合的水平床垫缝合皮肤。 注: 胸部少校和胸小肌不应缝合, 因为他们只是坦率地解剖和他们的解剖结构是完整的整个手术。</li>
</ol>4. 术后管理
<ol><li>每日两次通过皮下注射来管理丁丙诺啡 (3 毫克/千克), 以减少手术后48小时的术后疼痛11。</li>
	<li>手术后, 立即将小鼠保持在热垫上 (37 摄氏度), 直到完全恢复。将鼠标放回笼子后, 它完全恢复。</li>
	<li>在高温/高压灭菌器中杀菌使用的哈密尔顿注射器和30克注射器 (见步骤1.4 注)。在锋利的容器里收集消毒的30克注射器针。</li>
</ol>5.体内成像测量心脏荧光素酶表达的方法
<ol><li>在5天的腺病毒注射后, 准备一个新的库存溶液的荧光素在15毫克/毫升在 Dulbecco 的磷酸盐缓冲盐水 (DPBS)。通过0.2 µm 过滤器筛选解决方案。</li>
	<li>peritoneally 内注射荧光素溶液150毫克/千克体重的醒鼠。</li>
	<li>打开成像系统。</li>
	<li>成像系统自动自检后, 选择成像模式为 "发光", 并进行以下设置: 曝光: 自动;Binning: 8;FStop: 1;励磁: 块;发射: 打开。</li>
	<li>在荧光素注射液后10分钟, 用水合氯醛注射液麻醉小鼠 (300 毫克/千克体重)。确认足够的麻醉, 没有脚趾捏反应。</li>
	<li>将麻醉小鼠固定在俯卧部位的热成像台上, 将胸部对准摄像机。</li>
	<li>收集图像 (每只鼠标1图像), 并通过绘制相同的圆形测量区域 (ROI) 来量化信号的强度。</li>
	<li>在采集图像后, 牺牲小鼠并收集以下部分所述的组织。</li>
</ol>6. 收获组织
<ol><li>在病毒注射后的不同时间点 (图 1B), 麻醉小鼠与4% 异氟醚, 并通过鼻锥维持麻醉与2% 异氟醚, 并确保它在一个塑料平台上的俯卧位。</li>
	<li>在前颈部进行中线腹侧切口。通过缩回肩胛舌骨和胸锁乳突肌来暴露右颈总动脉。</li>
	<li>通过 transecting 正确的颈总动脉和排泄血液来牺牲老鼠。</li>
	<li>在腹部做一个中线腹侧切口。沿锁骨中线线切开肋笼两侧的肋骨, 并横断面膜片。</li>
	<li>举起剑突, 露出心脏。找到升主动脉, 轻轻地去除周围脂肪组织。</li>
	<li>Cannulate 主动脉和 retrogradely 灌注心脏与一个30克针连接到1毫升注射器填充0.5 毫升4°c 磷酸盐缓冲液 (PBS)。</li>
	<li>灌注后, 将心脏切除, 并将其分为左、右心室。切除肝脏、肺和脾脏。</li>
	<li>两次洗涤后, 冷 PBS (4 °c), 存储所有收集的组织分别在低温瓶中液氮。</li>
</ol>7. 蛋白质表达的测定
<ol><li>将蛋白酶抑制剂鸡尾酒加入到商业上可用的裂解缓冲液中, 以1:100 的比例制备均匀化缓冲剂。根据要处理的样本数计算总体积。将准备好的均匀缓冲保持在4摄氏度, 以供后续使用。</li>
	<li>从液氮罐中取出组织。在高精度鳞片上对组织进行称量 (每片组织大约60毫克)。</li>
	<li>将组织转移到一个新的1.5 毫升离心管。立即加入200µL 均匀缓冲和预换热器1.5 毫米钢球 (4 °c) 到离心管。</li>
	<li>将预换热器 (4 °c) 金属夹中的离心管固定在自动组织磨床上, 并以下列设置启动机器, 开始均匀化: 频率:70 赫兹;时间: 120s。</li>
	<li>均匀化后, 离心匀浆 (1.6万 x 克, 4 °c), 并小心转移到上清到一个新的1.5 毫升管与100µL 吸管。</li>
	<li>添加负载缓冲液 (5x) 到蛋白上清的比例为 1:4, 并变性蛋白质在100°c 5 分钟。</li>
	<li>在心脏和其他器官组织的蛋白质表达水平进一步监测的西方污点分析 (代表性的结果显示在图 2C和图 3) 根据协议, 先前描述的12。</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramyocardial+cell+delivery:+observations+in+murine+hearts.">Intramyocardial cell delivery: observations in murine hearts.</a> J Vis Exp. (83), e851064 (2014).</li></ol>

作者没有什么可透露的。

本报告展示了一种改进的方法, 心肌注射病毒载体的心脏基因操作, 这是修改了从心肌梗死的方法诱导的高等。13当前,在体内对特定基因功能的刻画最常涉及产生挖空或转基因小鼠3,14,15,16,17, 这是昂贵的、耗时的和劳力密集型的。或者, 通过系统或局部注射来传递基因载体或 siRNA 也被广泛应用于心血管研究的基因操作4,5,7。特别是, 在某些情况下, 心肌注射液不能替代心脏基因的操作: 当需要定点注射 (例如, 心肌梗死模型中的边界区注射)11;当需要持续时间限制基因操作时 (例如, 腺病毒注射)。结果表明, 改进后的心肌注射方法简单、省时、效率高。
协议中的关键步骤和疑难解答:
为了成功地执行本议定书, 应注意到几个关键步骤。在吸病毒之前, 汉密尔顿注射器内的空气和附针必须疏散, 否则注入心肌的空气可能引起局部心脏损伤甚至死亡。为了进一步避免这一问题, 准备就绪使用的哈密尔顿注射器填充适当的病毒体积不应放置在柱塞末端向下, 因为这可能自发吸入空气的严重性, 金属柱塞。麻醉应仔细监测, 因为深度麻醉可能会延迟手术后的恢复, 严重的深度麻醉可能导致死亡。经过充分的麻醉后, 心脏不应该被外力外化, 因为这可能导致严重的肺损伤。事实上, 适当的心外化只需要温柔的心, 任何抵抗都可能表明向不正确的方向前进。
技术的局限性:
这项技术的取消插管, 减少了程序所需的时间, 建议心肌注射程序应在一个相对有限的时间窗口内完成, 以避免死亡。根据我们的经验, 心脏不应该被外部化超过三十年代, 因为这增加了死亡率和减慢手术后恢复。因此, 建议使用插管在这里描述的协议的第一次尝试。另一个限制是这种方法提供的病毒载体的不均匀分布, 这也存在于传统的心肌注射方法7中。此外, 这里描述的方法更有用的心脏特异基因操作在未受伤的心脏, 可以随后建立不同的心脏病模型18,19,20, 21,22,23。然而, 使用目前的方法来向受伤的心脏, 如梗死心脏提供药物可能是有限的, 因为这些老鼠可能不容忍的程序。
对于现有方法的意义:
常规的心肌注射需要插管和机械通气24, 并使得很难找到注射部位由于快速的鼠标心跳。这些问题大大延长了操作时间13, 从而增加了时间延迟带来的变化。这里提出的改进技术是快速的, 并允许精确的注射地点, 手动保护外部心脏;总的来说, 显著 potentiating 随后的研究。此外, 消除插管和机械通气使修改的方法几乎可以进入任何实验室。

心脏病是全球发病率和死亡率的主要原因1,2。缺乏有效的治疗策略, 包括心肌梗死和心力衰竭等危及生命的心脏疾病, 吸引了对基础 pathomechanisms 的深入探索, 并确定了新的治疗方案3。对于这些科学探索, 心脏特异基因操作被广泛使用4,5。心脏基因操纵可以通过基因组编辑使用强大的转录激活器样效应核酸酶 (塔伦) 和聚集定期 interspaced 短回文重复 (CRISPR)/CRISPR 相关蛋白 9 (Cas9) 工具, 或通过交付异位基因材料 (例如, 携带感兴趣的蛋白质编码基因的病毒载体)6。虽然基因组编辑允许精确和时空基因组修改的活鼠, 它仍然是一个耗时和劳动密集型的做法6。或者, 由病毒载体或小干扰 RNA (siRNA) 复杂传递的心脏特异基因操作通常执行6。
病毒载体传递到成年鼠心脏是通过大致两个策略: 系统性或局部注射。系统性注射 cardiotropic 血清自动增值服务如 AAV9 是无创性的心脏特异基因操作7。然而, 这种方法需要一个相对较高的载体, 以有效的传导和基因表达, 并可能导致显着转导的 nontarget 器官, 如肌肉和肝脏的7。局部病毒注射是通过心肌注射或冠状动脉内传递7实现的。与心肌注射液相比, 冠状动脉内分娩可导致心脏内更均匀的病毒分布。然而, 这一技术的缺点是快速洗出的病毒载体的系统循环和转导的 nontarget 器官8, 以及它的要求设备的压力测量在操作过程中。相比之下, 心肌注射液能使心肌中的病毒保留更好, 以及特定部位的分娩, 但它无法均匀分布病毒载体7。对小动物来说, 冠状动脉内分娩在技术上很难执行, 而系统性 AAV9 注射和心肌注射则更常用4,5,7。虽然全身注射是容易执行, 常规心肌注射需要机械通气和开胸, 造成广泛的组织损伤, 是费时。
在本报告中, 我们描述了一种简单、节省时间和高效的心肌注射方法。注射荧光素酶和 vdr 的腺病毒, 或 shRNA 靶向 vdr, 被注入以操纵心脏基因表达。一旦掌握, 这种方法可以用于基因操作, 以及注射细胞或其他材料的老鼠心脏。

<table><tbody><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Laminar flow sterile hood</td><td>Fengshi Animal Experimental&amp;nbsp; Equipment Techonology Co., Ltd. (Soochow, China)</td><td>FS-CJ-2F</td><td></td></tr><tr><td>Centrifuge</td><td>Thermo Scientific (Waltham, USA)</td><td>75005282</td><td></td></tr><tr><td>Tissue grinding machine</td><td>Scientz Biotechnology Co., Ltd. (Ningbo, China)</td><td>Scientz-48</td><td></td></tr><tr><td>High temperature/high pressure sterilizer</td><td>Hirayama (Saitama, Japan)</td><td>HVE-50</td><td></td></tr><tr><td>Isoflurane vaporizer&amp;nbsp;</td><td>Matrix (Orchard Park, USA)</td><td>VIP3000</td><td></td></tr><tr><td>IVIS&amp;nbsp; Lumina III imaging system</td><td>PerkinElmer (Waltham, USA)</td><td>CLS136334</td><td></td></tr><tr><td>Precision&amp;nbsp;balance</td><td>Sartorius (G&amp;ouml;ttingen, Germany)</td><td>28091873</td><td></td></tr><tr><td>&lt;strong&gt;Instruments&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Eppendorf pipette (100 &amp;micro;L)</td><td>Eppendorf (Westbury, USA)</td><td>&amp;nbsp;4920000059</td><td></td></tr><tr><td>Eppendorf pipette (10 &amp;micro;L)</td><td>Eppendorf (Westbury, USA)</td><td>&amp;nbsp;4920000113</td><td></td></tr><tr><td>Forceps</td><td>Shanghai Medical Instruments (Group) Ltd., Corp.&amp;nbsp;</td><td>JD4020</td><td>Curved tip</td></tr><tr><td>Hamilton syringe</td><td>Hamilton (Nevada, USA)</td><td>80501</td><td>Volume 50 &amp;mu;L</td></tr><tr><td>Micro-mosquito hemostat</td><td>F.S.T (Foster City, USA)</td><td>13011-12</td><td>Curved, tip width 1.3mm</td></tr><tr><td>Needle holder&amp;nbsp;</td><td>Shanghai Medical Instruments (Group) Ltd., Corp. (Shanghai, China)</td><td>J32110</td><td></td></tr><tr><td>Surgical scissors</td><td>F.S.T (Foster City, USA)</td><td>14002-12</td><td></td></tr><tr><td>1-mL Syringe</td><td>WeiGao Group Medical Polymer Co.,Ltd. (ShangDong, China)</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Materials and reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-GAPDH antibody</td><td>CST (Danvers,&amp;nbsp; USA)</td><td>#2118</td><td></td></tr><tr><td>Anti-Luciferase antibody</td><td>Abcam (Cambridge, UK)</td><td>ab187340</td><td></td></tr><tr><td>Anti-rabbit IgG</td><td>CST (Danvers,&amp;nbsp; USA)</td><td>&amp;nbsp;#7074</td><td></td></tr><tr><td>Anti-VDR antibody</td><td>Abcam (Cambridge, UK)</td><td>&amp;nbsp;ab109234</td><td></td></tr><tr><td>Buprenorphine</td><td>Thermo Scientific (Waltham, USA)</td><td>PA175056</td><td></td></tr><tr><td>Chloralic hydras</td><td>LingFeng Chemical (ShangHai, China)</td><td></td><td></td></tr><tr><td>Cryogenic Vials</td><td>Thermo Scientific (Waltham, USA)</td><td>375418</td><td>1.8 mL&amp;nbsp;</td></tr><tr><td>Depilatory cream</td><td>Veet (Shanghai, China)</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline&amp;nbsp;</td><td>Gibco (Grand Island,&amp;nbsp; USA)</td><td>14040133</td><td></td></tr><tr><td>Entoiodine</td><td>LiKang (Shanghai, China)</td><td>310132</td><td></td></tr><tr><td>EP tube</td><td>Sarstedt (Newton, USA)</td><td>PCR001</td><td></td></tr><tr><td>Filter</td><td>Millipore (Bedford, USA)</td><td></td><td>Pore size 0.2 &amp;micro;m&amp;nbsp;</td></tr><tr><td>Isoflurane</td><td>Yipin Pharmaceutical Company (Hebei, China)</td><td></td><td></td></tr><tr><td>Luciferin</td><td>Promega (Madison, USA)</td><td>P1041</td><td></td></tr><tr><td>Lysis buffer for western&amp;nbsp; blot</td><td>Beyotime (Shanghai, China)</td><td>P0013J</td><td>Without inhibitors</td></tr><tr><td>Ophthalmic cream</td><td>Apex Laboratories ( Melbourne, Australia))</td><td></td><td></td></tr><tr><td>PBS</td><td>Gibco (Grand Island,&amp;nbsp; USA)</td><td>10010023</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Thermo Scientific (Waltham, USA)</td><td>78438</td><td></td></tr><tr><td>5-0 silk suture</td><td>Shanghai Medical Instruments (Group) Ltd., Corp. (Shanghai, China)</td><td></td><td></td></tr><tr><td>Steel ball</td><td>Scientz Biotechnology Co., Ltd. (Ningbo, China)</td><td></td><td>Width 1.5 mm</td></tr><tr><td>Syringe needle</td><td>Kindly Medical Devices Co., Ltd. (Zhejiang, China)</td><td></td><td>30 gauge&amp;nbsp;</td></tr><tr><td>Warm mat</td><td>Warmtact Electrical Heating Technology Co., Ltd. (Guangdong, China )</td><td>NF-GNCW</td><td></td></tr></tbody></table>

a simple and efficient method for in vivo cardiac-specific gene manipulation by intramyocardial injection in mice

特别是在心脏的基因操作明显事实心脏疾病 pathomechanisms 的调查和他们的治疗潜力。体内心脏特异基因的传递通常是通过系统性或局部分娩来实现的。应用重组腺相关病毒 9 (AAV9), 采用尾静脉全身注射治疗心脏基因表达简便、有效。然而, 这种方法需要较高的载体, 有效传导, 并可能导致 nontarget 器官基因转导。在这里, 我们描述了一个简单的, 有效的, 节省时间的方法, 心肌注射液的体内心脏特异基因操作的小鼠。在麻醉 (没有通气) 下, 胸部主要和次要肌肉被坦率地解剖, 并且小鼠心脏通过手工外在化快速地暴露通过小切口在第四肋间空间。随后, 腺病毒的编码荧光素酶 (卢克) 和维生素 D 受体 (vdr), 或短发夹 RNA (shRNA) 靶向 VDR, 注射了汉密尔顿注射器进入心肌。随后的体内成像表明, 荧光素酶成功地抗原心脏。此外, 西方印迹分析证实了成功的过度表达或沉默的 VDR 在小鼠心脏。一旦掌握, 这种技术可以用于基因操作, 以及注射细胞或其他材料, 如磁性在鼠标心脏。

实验协议和报告方法的一些关键步骤显示在图 1中。5天后, 在心肌注射腺病毒荧光素酶 (), 在体内成像在对卢克注射小鼠表明, 显着的强力表达的荧光素酶特别在心脏 (图2A, B), 这是由西方印迹分析 (图 2C) 证实, 表明没有 nontarget 器官转导。相比之下, 在控制小鼠中没有发现荧光素酶表达。根据荧光素酶的成功过度表达, 西方印迹分析表明, 在注射腺病毒的小鼠左心室中, vdr 的表达明显增加 (如 vdr) (图 3A)。此外, shVDR 注射液显著减少左心室的 VDR 表达 (图 3B)。与此相反, 在右心室, shVDR 注射小鼠或注射小鼠 (图 3C, D) 中, vdr 表达没有明显改变, 因为腺病毒只注入左心室心肌。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57074/57074fig1.jpg"> 图 1.小鼠心肌注射和基因表达检测协议的模式.(A) 图示显示左心室心肌中有三个注射点。(B) 提示病毒注射后小鼠心脏基因表达检测的协议。荧光素酶: 腺病毒荧光素编码;AdVDR: 腺病毒编码 VDR;AdshVDR, 腺病毒编码 shRNA 靶向 VDR。(C) 代表图像, 显示修改后的鼠标心肌注射方法的多个步骤。a.通过商业上可用的脱毛膏去除毛皮。b. 3 度聚维酮碘磨砂手术部位的灭菌。c.用无菌悬垂覆盖手术部位。d. 0.5 厘米皮肤切口沿线连接剑突和腋窝。e.用镊子和微蚊子止血钝解剖胸大肌和胸小肌肉。f.心脏的外化。g.通过哈密尔顿注射器将30µL 腺病毒溶液注入左心室心肌。h-i.用5-0 丝缝线缝合的钱包线缝合皮肤。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57074/57074fig1large.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/57074/57074fig2.jpg"> 图 2.检测心脏中的荧光素酶表达式.(A) 影像系统采集的图像, 显示在注射后5天内, 在控制小鼠和注射腺病毒荧光素酶的小鼠体内, 心脏的发光信号。(B) 不同组的发光信号强度 (n = 3) 受t测试的统计分析。**p < 0.01 与控制鼠标。(C) 西方印迹分析结果显示, 在腺病毒注射后5天内, 表明组的心、肺、肝和脾中的荧光素酶蛋白水平 (n = 3)。由于荧光素酶的表达仅在对卢克注射小鼠心脏的检测, 没有进行统计分析。编码荧光素酶的腺病毒。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57074/57074fig2large.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/57074/57074fig3.jpg"> 图 3.检测心脏中的 VDR 表达式.(A) 顶部面板: 在0天、5天、7天和在注射后14天, 显示在左心室 (腺病毒注射) 的 VDR 水平的西方印迹带。底部板: 对 VDR 表达水平的半定量分析在不同的小组 (n = 4 每时间点)。结果被规范化对 GAPDH 并且转换了到折叠变动相对于0天。对方差 (方差分析) 后的 Bonferroni 后检验 (等方差假设) 或 Tamhane 后检验 (不假设的等方差) 进行了统计分析。*p < 0.05 或 **p < 0.01 vs 0 天。(B) 顶部面板: 在0天、3天、5天和 shVDR 注射液后7天, 在左心室显示 VDR 水平的西方印迹带。底部板: 对 VDR 表达水平的半定量分析在不同的小组 (n = 4 每时间点)。*p < 0.05 vs 0 天。(C) 顶部面板: 在0天、5天、7天和在注射后14天内, 显示在右心室 (腺病毒未注射) 的 VDR 水平的西方印迹带。底部板: 对 VDR 表达水平的半定量分析在不同的小组 (n = 4 每时间点)。(D) 顶部面板: 在0天、3天、5天和 shVDR 注射液后7天, 在右心室显示 VDR 水平的西方印迹带。底部板: 对 VDR 表达水平的半定量分析在不同的小组 (n = 4 每时间点)。<a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57074/57074fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice at JoVE.com

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

在这里, 我们提出了一个小鼠心脏特异基因操作的协议。在麻醉下, 小鼠心脏通过第四肋间空间被外部化。随后, 将特定基因的腺病毒注射进心肌, 然后通过体内成像和西方印迹分析对蛋白质表达进行测量。

一种简便、有效的心肌注射小鼠心脏特异基因操作方法--体内

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

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Universidad San Sebastian

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JoVE Journal

Biology

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

15.8K 次观看.  The First Affiliated Hospital of Bengbu Medical College. 在这里, 我们提出了一个小鼠心脏特异基因操作的协议。在麻醉下, 小鼠心脏通过第四肋间空间被外部化。随后, 将特定基因的腺病毒注射进心肌, 然后通过体内成像和西方印迹分析对蛋白质表达进行测量。

视频: A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

结扎冠状动脉内注射在小鼠模型梗死

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

科研

医学

Delivery of Modified mRNA in a Myocardial Infarction Mouse Model

Myocardial infarction (MI) is a leading cause of morbidity and mortality in the Western world. In the past decade, gene therapy has become a promising treatment option for heart disease, owing to its efficiency and exceptional therapeutic effects. In an effort to repair the damaged tissue post-MI, various studies have employed DNA-based or viral gene therapy but have faced considerable hurdles due to the poor and uncontrolled expression of the delivered genes, edema, arrhythmia, and cardiac hypertrophy. Synthetic modified mRNA (modRNA) presents a novel gene therapy approach that offers high, transient, safe, nonimmunogenic, and controlled mRNA delivery to the heart tissue without any risk of genomic integration. Due to these remarkable characteristics combined with its bell-shaped pharmacokinetics in the heart, modRNA has become an attractive approach for the treatment of heart disease. However, to increase its effectiveness in vivo, a consistent and reliable delivery method needs to be followed. Hence, to maximize modRNA delivery efficiency and yield consistency in modRNA use for in vivo applications, an optimized method of preparation and delivery of modRNA intracardiac injection in a mouse MI model is presented. This protocol will make modRNA delivery more accessible for basic and translational research.

This protocol presents a simple and coherent way to transiently upregulate a gene of interest using modRNA after myocardial infarction in mice.

在心肌梗死小鼠模型中传递修正的mRNA

Myocardial infarction (MI) is a leading cause of morbidity and mortality in the Western world. In the past decade, gene therapy has become a promising ...

遗传学

Intramyocardial Cell Delivery: Observations in Murine Hearts

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue revascularization and cell survival. In addition, further observations revealed that specific stem cells, such as cardiac stem cells, mesenchymal stem cells and cardiospheres have the ability to integrate within the surrounding myocardium by differentiating into cardiomyocytes, smooth muscle cells and endothelial cells.
Here, we present the materials and methods to reliably deliver noncontractile cells into the left ventricular wall of immunodepleted mice. The salient steps of this microsurgical procedure involve anesthesia and analgesia injection, intratracheal intubation, incision to open the chest and expose the heart and delivery of cells by a sterile 30-gauge needle and a precision microliter syringe.
Tissue processing consisting of heart harvesting, embedding, sectioning and histological staining showed that intramyocardial cell injection produced a small damage in the epicardial area, as well as in the ventricular wall. Noncontractile cells were retained into the myocardial wall of immunocompromised mice and were surrounded by a layer of fibrotic tissue, likely to protect from cardiac pressure and mechanical load.

Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.

心肌内细胞交货：观察在小鼠心脏

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

基于微注射的免疫胚胎心肌细胞体内植入系统

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

发育生物学

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

超声引导下经皮内注射小鼠

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

小鼠心脏中基于电穿孔的心肌细胞转基因

An Efficient Transgenesis Approach for Gene Delivery in the Mouse Embryonic Heart

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, and fate of these progenitors are vital for the proper development of this organ. The molecular mechanisms that govern the morphogenesis of the heart are essential for understanding the pathogenesis of congenital heart diseases and embryonic cardiac regeneration. Classical approaches to investigate these mechanisms employed the generation of transgenic mice to assess the function of specific genes during cardiac development. However, mouse transgenesis is a complex, time-consuming process that often cannot be performed to assess the role of specific genes during heart development. To address this, we have developed a protocol for efficient electroporation and culture of mouse embryonic hearts, enabling transient transgenesis to rapidly assess the effect of gain- or loss-of-function of genes involved in cardiac development. Using this methodology, we successfully overexpressed Meis1 in the embryonic heart, with a preference for epicardial cell transfection, demonstrating the capabilities of the technique.

作者聚焦:用于快速基因筛选的心肌细胞转基因

This protocol presents a detailed methodological framework for electroporation-based transgenesis of cardiac cells in developing mouse hearts. The video assets provided here will facilitate learning of this versatile technique.

一种在小鼠胚胎心脏中进行基因递送的高效转基因方法

The mammalian heart is a complex organ formed during development via highly diverse populations of progenitor cells. The origin, timing of recruitment, ...

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

经皮超声心动图引导心肌注射液与大鼠前体细胞的临床研究

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...

Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and living organisms from a wide variety of species. The CRISPR technology has also been explored as novel therapeutics for a number of human diseases. Proof-of-concept data are highly encouraging as exemplified by recent studies that demonstrate the feasibility and efficacy of gene editing-based therapeutic approach for Duchenne muscular dystrophy (DMD) using a murine model. In particular, intravenous and intraperitoneal injection of the recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) has enabled efficient cardiac delivery of the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and two guide RNAs (gRNA) to delete a genomic region with a mutant codon in exon 23 of mouse Dmd gene. This same approach can also be used to knock out the gene-of-interest and study their cardiac function in postnatal mice when the gRNA is designed to target the coding region of the gene. In this protocol, we show in detail how to engineer rAAVrh.74-CRISPR vector and how to achieve highly efficient cardiac delivery in neonatal mice.

Here we provide a detailed protocol to carry out in vivo cardiac gene editing in mice using recombinant Adeno-Associated Virus(rAAV)-mediated delivery of CRISPR. This protocol offers a promising therapeutic strategy to treat dystrophic cardiomyopathy in Duchenne muscular dystrophy and can be used to generate cardiac-specific knockout in postnatal mice.

腺相关病毒介导的 CRISPR 在小鼠心脏基因编辑中的传递

The clustered, regularly interspaced, short, palindromic repeat (CRISPR) system has greatly facilitated genome engineering in both cultured cells and ...

一种简便、有效的心肌注射小鼠心脏特异基因操作方法--体内

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此视频中的章节

结扎冠状动脉内注射在小鼠模型梗死

在心肌梗死小鼠模型中传递修正的mRNA

心肌内细胞交货：观察在小鼠心脏

基于微注射的免疫胚胎心肌细胞体内植入系统

超声引导下经皮内注射小鼠

一种在小鼠胚胎心脏中进行基因递送的高效转基因方法

一种在小鼠胚胎心脏中进行基因递送的高效转基因方法

一种在小鼠胚胎心脏中进行基因递送的高效转基因方法

经皮超声心动图引导心肌注射液与大鼠前体细胞的临床研究

腺相关病毒介导的 CRISPR 在小鼠心脏基因编辑中的传递