Name: in vivo nanovector delivery of a heart-specific microrna-sponge
Uploaded: 2018-06-15T15:00:00.000+00:00
Duration: 9 min 53 s
Description: Watch this Scientific Journal Video about In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge at JoVE.com

我们感谢彭博公共卫生学院生物化学与分子生物学系的安东尼 k.l. 梁, 他在设计 miR-181-sponge 结构方面的技术帮助。我们还感谢分子和比较病理部的宝琳娜 Sysa 和凯瑟琳 Gabrielson, 约翰霍普金斯医疗机构的技术援助, 通过体内成像的 miRNA 海绵分娩。
这项工作得到了 NIH、HL39752 (查尔斯 Steenbergen) 的赠款和美国心脏协会 14SDG18890049 (Samarjit Das) 的科学家发展补助金的支持。在辛辛那提儿童医院, 杰弗里 d. Molkentin 慷慨地提供了鼠心特异性启动子。

所有实验程序均经约翰霍普金斯大学机构动物护理和使用委员会批准。
1. 海绵设计
<ol><li>
MicroRNA 绑定 3 ' UTR 注: miRNA 函数通过特定的基配对与部分互补点在其目标基因的 3 ' 未翻译区域 (UTR) 中的相互作用 (为全面审查, 见巴特尔)15。<ol>
<li>设计 miRNA 表达抑制剂作为寡核苷酸, 对 miRNA 序列具有显著的互补性。通过广泛的基配对将 miRNA 在寡核苷酸复合体中, 以阻止 miRNA 功能。</li>
	</ol>
</li>
	<li>
miRNA 海绵的设计
	<ol>
<li>设计 tandemly 阵列部分互补 miRNA 序列, 包括一个凸起的位置通常由 Argonaute 2。凸起位于成熟 miRNA 序列的核苷酸 9-12, 以防止任何 RNA 干扰类型的分裂和海绵的降解。</li>
		<li>如果需要, 设计诱饵目标序列绑定到所有 miR-181 家庭成员。在检查和比较 miR-181 家庭 miRNA 序列, 发现一个共同的序列在 5 ' 边的凸起包含8个连续核苷酸, 或者发现一个序列共同的4个 miR-181 家庭成员在 3 ' 边膨胀的另外8连续核苷酸。这2个序列分别表示左和右半绑定站点。 注: 诱饵序列是由 GGA 间隔分隔的左右半绑定站点的串联数组。将 5 '-端序列和 3 ' 端序列 (在步骤1.2.2 中描述) 连接在一起, 其设计为在退火到 miRNA 时创建凸起 (如步骤1.2.1 中所述)。重复这 1 miRNA 绑定站点10x 在最后的和平号-海绵结构。</li>
	</ol>
</li>
	<li>
其他 miRNA 海绵的注意事项 注意: 为了将海绵序列复制到接收者表达式向量中, 必须在序列的每一端包含限制核酸酶识别站点。在硅消化分析中, 应使用所选择的限制酶和在下游克隆应用中使用的其他限制酶来确定海绵序列的非特异裂解。<ol>
<li>在合成 3 ' UTR 的 5 ' 端添加一个双停止密码子 (TAA TAA), 这样 miR-181-sponge 序列不是增强的绿色荧光蛋白 (EGFP) 编码序列的一部分。</li>
	</ol>
</li>
	<li>
克隆海绵 DNA 的合成
	<ol>
<li>使用 DNA 合成器或利用商业可用的来源, 以合成最终 miRNA 海绵序列与限制核酸酶识别网站到位, 克隆。海绵被运送到一种质粒上, 可以包含可选择的标记, 用于放大细菌。此外, 海绵可以通过聚合酶链反应 (PCR) 来进行克隆, 或者使用步骤1.3 中描述的限制点 (也见步骤 3.1) 来简单地丢弃捐赠向量。</li>
	</ol>
</li>
</ol>2. 克隆受体载体, 包括组织特定的启动子
注: 使用 pEGFP-C1 向量作为 miRNA 海绵表达盒的接收向量。pEGFP-C1 向量包含一个 EGFP 由巨细胞病毒 (CMV) 启动子驱动的阅读框架。巨细胞病毒启动子是组成性活跃在哺乳类。如果需要组织特定的启动子, 则可以通过消化 AseI 和 NheI 酶来去除巨细胞病毒启动子 (见步骤 2.1)。该质粒包含一个广泛的多克隆站点 (MCS) 和卡那霉素 (菅直人) 和新霉素 (G418) 标记的细菌选择和一个稳定的表达在哺乳动物细胞, 分别。
<ol><li>
从 pEGFP-C1 中去除巨细胞病毒启动子
	<ol>
<li>制作50的消化反应 ul, 其中含有1x 商业上可用的消化缓冲剂, 所用的限制酶的制造, 5 微克的质粒 DNA (在水中), 5 ul 每 AseI 和 NheI 酶。将所有组件添加到离心管中, 然后轻轻地将其混合。十年代在离心中旋转管子以收集底部的反应。在37摄氏度的水浴中孵化反应15分钟。</li>
		<li>纯化经消化的线性化质粒。添加5x 列绑定缓冲区的反应量 (用于制造 dna 纯化柱的适当绑定缓冲区的专有组合), 并按照制造商描述的 dna 纯化柱纯化反应。洗脱与 25 ul 洗脱缓冲 (水) 添加仔细到中心的专栏。</li>
		<li>凝胶纯化0.5% 琼脂糖凝胶上的消化质粒, 如下所示: 在洗脱质粒上添加5的负载缓冲 (50% glycerol-3x 加载染料) ul。在0.5% 琼脂糖凝胶上将整个样品装入1井。包括一个单独的车道与500吴未切割的载体。运行 30-40 分钟, 直到达到适当的分离切割和未切割的质粒。</li>
		<li>对线性化质粒进行纯化: 将琼脂糖凝胶中的 DNA 可视化为 UV 光盒。用干净的剃刀刀片, 仔细地使用与线性质粒对应的带。 注: 线性质粒应低于未切割的质粒, 在大约 4.2 kb 时将显示为单波段。该切除的巨细胞病毒启动子将出现作为一个光带在大约500核苷酸 (nt)。</li>
		<li>利用商业上可用的试剂盒从凝胶切片中提取 dna, 并洗脱 25 ul 的水柱上的 dna。</li>
	</ol>
</li>
	<li>
将心脏特定的启动子克隆到 pEGFP-C1 中 注: 阿尔法肌球蛋白重链 (α MHC) 启动子以前的特点和证明, 以直接的强健水平的心脏限制表达, 如以下 Molkentin, 乔布, 和锦马16。接下来的部分描述了如何放大克隆的向量的启动子。<ol>
<li>克隆的α MHC 启动子之间的 +420 至-2934 之间的转录起始点到 p40 质粒16首先设计 pcr 底漆, 侧面这个区域, 然后质粒作为 pcr 模板。使用 PCR 引物放大启动子区域以前描述的5。正向和反向 PCR 引物包含重叠序列的 pEGFP-C1 (步骤 2.1) 的消化结束, 以允许融合定向克隆。请参阅融合手册, 以全面解释融合克隆的底漆设计。底漆序列在表 1中找到。</li>
		<li>准备一个 50 ul PCR 反应, 其中包含1微米的每一个底漆和 10 ng 的模板 DNA。根据所选 pcr 试剂的制作, 添加适当数量的 pcr 酶和 dNTPs。在标准 DNA 循环块上进行 PCR。执行3分钟98°c 变性步骤, 其次为5、30和120s 的35个变性、退火和延伸周期分别。包括最后延长10分钟在72°c。</li>
		<li>清理 PCR 和凝胶萃取。在 pcr 循环完成后, 将柱状结合缓冲器的反应体积加5x 到 pcr 反应中, 并按照制造商描述的 DNA 纯化柱纯化。洗脱与 25 ul 的洗脱缓冲 (水) 的混合物, 小心地添加到柱的中心。<ol>
<li>添加5的负载缓冲 ul [50% 甘油 (3x) 加载染料] 到洗脱质粒。在1% 琼脂糖凝胶上将整个样品装入1井。运行 30-40 分钟;如上文所述, 可视化和消费 PCR 产品 (步骤 2.1.5)。</li>
		</ol>
</li>
		<li>结扎α MHC 启动子与 pEGFP-C1 如下: 添加 10 ul 结扎反应, 包括1x 商用结扎缓冲, 2 ul 的纯化 PCR, 1 ul 的线性化向量。执行结扎在50°c 15 分钟, 然后冷却它在冰上。</li>
		<li>执行细菌转化如下: 染 50 ul 的合格细胞与 2.5 ul 的融合结扎混合通过添加成分。把离心管放在冰上20分钟, 放在42摄氏度的水浴中, 四十年代, 然后把管子放在冰上2分钟。<ol>
<li>转换后, 添加 350 ul 的 SOC 介质和生长细胞在37°c 45 分钟. 板 200 ul 在 LB 板上与菅直人. 执行菌落 PCR 鉴定含有α MHC 启动子结扎的抗菅细胞。 注: 筛选底漆的设计, 以 PCR 横跨结扎连接。</li>
		</ol>
</li>
		<li>用标准的微型预制备质粒纯化协议, 以制造商描述的方法, 在 LB-坎汤中扩增 PCR 阳性克隆。</li>
	</ol>
</li>
</ol>3. 海绵的克隆
<ol><li>
摘要收件人向量
	<ol>
<li>用α-MHC-pEGFP 质粒线性克隆 EcoRI 和 KpnI, 并纯化上述凝胶 (步骤 2.1)。</li>
	</ol>
</li>
	<li>
PCR 放大和消化海绵 DNA
	<ol>
<li>使用 miR-181-sponge 和相应的 284 nt 长的争夺航运载体作为 PCR 模板。使用先前描述的 PCR 引物5放大海绵区域。注意, 正向和反向 PCR 引物含有限制酶序列, 允许结扎成α-MHC-pEGFP-C1。执行 PCR 如上文所述 (步骤 2.2.2)。在消化前纯化 PCR 产物 (步骤 2.2.3), 并洗脱水进行消化。对于底漆序列, 见表 1.
</li>
		<li>消化海绵 DNA 进行结扎。50 ul 消化反应包括1x 消化缓冲, 凝胶纯化 PCR 反应从步3.2.1 和 5 ul EcoRI 和 KpnI 酵素。将所有组件添加到离心管中, 然后轻轻地将其混合。十年代在离心中旋转管子以收集底部的反应。在37摄氏度的水浴中孵化反应15分钟。</li>
		<li>使用 pcr 清理柱纯化消化后的 pcr 海绵 (步骤 2.2.1)。</li>
	</ol>
</li>
	<li>
结扎 miR-181-sponge 成α-MHC-pEGFP C1 向量
	<ol>
<li>建立 21 ul 结扎反应, 其中包含1x 结扎缓冲, 3 ul 的消化和纯化 miR-181-sponge PCR, 1 ul 的线性α MHC-pEGFP C1 向量, 1x dna 缓冲, 1 ul 的 dna 连接酶。在室温下进行结扎5分钟, 然后放在冰上。</li>
		<li>50 ul 的 XL1-blue 主管细胞与 2 ul 的结扎组合。使用以上所述的转换和电镀协议 (步骤 2.2.5)。进行菌落 PCR 鉴定含有正确的α-MHC-pEGFP-181 海绵序列的耐药性细菌。设计筛选引物的 PCR 横跨结扎连接。见表 1为底漆序列。</li>
		<li>放大和序列的向量。用标准的微型预制备质粒纯化协议, 在 LB 汤中扩增 PCR 阳性克隆, 并将其纯化成粒。进行 dna 测序以验证表达所需 dna 序列的质粒。</li>
	</ol>
</li>
</ol>4. 生成稳定的 H9c2 miR-181-sponge 表达细胞
<ol><li>
H9c2 细胞的生长与维持
	<ol>
<li>培养 H9c2 细胞在 Dulbecco 的改良鹰的培养基 (DMEM) 含有胎牛血清 (血清) 到最后浓度为10%。亚文化细胞使用标准的协议, 并在5% 二氧化碳37摄氏度增长。</li>
	</ol>
</li>
	<li>
H9c2 α-pEGFP-C1-181-海绵表达细胞的转染与选择
	<ol>
<li>对 H9c2 细胞进行电穿孔, 以取得最佳效果。染大鼠成肌细胞 (H9c2), 用一只爬行海绵 (一个 284 nt 长的争夺序列取代 miR-181-sponge-sequence) 或α-MHC-pEGFP 和平号-181 海绵结构与 electroporator。<ol>
<li>简单地, 染 4x10-5细胞与2微克的质粒 DNA (在5µL 的 H2O) 和100µL 的解决方案兼容的电穿孔装置。使用 DS-120 的电穿孔程序染 H9c2 细胞。</li>
			<li>室温下在试管中孵育转染细胞10分钟。将500µL DMEM 直接添加到试管。将总试管内容轻轻地转移到6井板的井中。</li>
		</ol>
</li>
		<li>选择荧光蛋白阳性细胞后, 48 小时后转染的外地资产管制。仅板绿色 GFP 表达的细胞入6井板材与增加新霉素 (G418) (400 ng/毫升) 到完全成长媒介。 注意: 在 G418 选择之前, 应建立一个终止曲线, 以确定选定内容所需的新霉素量。对于 H9c2 细胞, 400 G418 的80% 的非质粒转染 H9c2 细胞死亡率约为24小时后, 被认为是这些细胞所期望的 G418 工作浓度。在 G418 维持16天后, 细胞线被认为是稳定的。</li>
		<li>用 GFP 的表达作为海绵阳性选择的标记, 对 G418 稳定细胞进行分类。通过流动排序, 在96井板的每一个井中种 5-10 个细胞。将单克隆细胞从96井板扩展到24井板上的多个通道。使用冷冻储存的细胞从通道 3 (P3) 细胞作为后续实验的出发点。</li>
		<li>在 P5 和 P10 之间执行所有基于细胞线的低通道细胞实验。</li>
	</ol>
</li>
</ol>5. 海绵纳米微粒的合成和纯化 (miR-181-sponge 纳米粒子)
<ol><li>将阳离子 amphiphile (DOTAP) 和共脂、胆固醇和 DSPE 钉在 5:5: 0.1 毫米的比值中, 分别用一个玻璃瓶中的氯仿和甲醇混合物制备脂质体纳米颗粒。</li>
	<li>在真空下使用旋转蒸发器将有机溶剂去除 44-45 摄氏度, 其次是温和流动的无水分氮气。将剩余的脂质干膜保持在高真空下8小时。</li>
	<li>在真空干脂膜中加入5% 葡萄糖来制备混合物。让它过夜, 让这种混合物水合物的电影。在室温下将瓶子旋转2到3分钟, 以便在 45° C 水浴中偶尔摇动, 产生 multilamellar 泡。<ol>
<li>油脂实验 multilamellar 泡在冰浴中准备小 unilamellar 泡, 3-4 分钟, 直到可以观察到清晰度, 在100% 的工作周期和 25 W 输出功率。</li>
	</ol>
</li>
	<li>将 miR-181-sponge 序列或克隆成 pEGFP (α-MHC) 载体和脂质体的一组混合序列与1:3 电荷比基础, 形成 nanovector4。 注: 有正电荷的脂质体纳米粒子和带负电荷的粒 DNA 的静电复合物被称为 nanovector。</li>
</ol>6. 海绵纳米微粒的全身输送和体内效应的验证
<ol><li>
海绵纳米颗粒在大鼠体内的系统传递
	<ol>
<li>使用大 (SD) 大鼠。静脉注射 miR-181-sponge nanovector 的大鼠, 或通过尾静脉 nanovector。使用 SD 雄性大鼠, 其体重约为200克。</li>
		<li>使用4毫克 nanovector/千克体重的剂量进行尾静脉注射, 并在2周内使用6尾静脉注射方案进行重复。在重复 nanovector 交付后, 允许1周 (天 15-21) 在体内表达载体。</li>
		<li>通过在 nanovector 交付之前、期间和之后对 GFP 信号进行成像, 确认优化。利用层析成像软件对 GFP 信号进行分析, 生成半定量数据。</li>
	</ol>
</li>
	<li>
miR-181-sponge 对体内miR-181 抑制作用的验证
	<ol>
<li>执行西方印迹, 以证明在心脏组织的功能 miR-181-sponge 的表达。 注: 本研究对 mt-COX1 表达进行了检查。</li>
	</ol>
</li>
	<li>
心脏体内miR-181-sponge 表达的功能后果
	<ol>
<li>在 nanovector 分娩前后进行二维、M 型和多普勒超声心动图分析心脏功能和形态学改变。为了更好的扫描能力, 在啮齿动物的心脏, 使用一个超声系统配备 15 MHz 线性阵列传感器。</li>
		<li>麻醉可能影响心脏收缩力;因此, 在超声心动图过程中, 执行 miR-181-sponge nanovector 或争夺 nanovector 的动物, 并无任何麻醉试剂。请见 Das,等等。4进一步澄清。</li>
	</ol>
</li>
</ol>

<ol>
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作者没有什么可透露的。

本文介绍了 miRNA 海绵的设计和合成, 并论证了海绵组织特异表达是抑制组织特异 miRNA 家族表达的有力工具。
我们已经证明, 一个 miR-181 的家庭靶向海绵可以克隆成表达质粒与心脏特定的启动子。该质粒可以有效地包装成 nanovector 颗粒, 用于在体外和体内使用电穿孔或尾静脉注射, 分别 (图 1)。miR-181 海绵可以抑制 miR-181 家族的心脏特异表达, 并可能影响 miR-181 靶基因的表达 (图 2)。质粒中的 GFP 是一个额外的优势, 它可以可视化的传递和 nanovector 的表达谱, 而不牺牲动物。
简单地说, miRNA 海绵由一系列 miRNA 反义序列组成, 其作用是作为诱饵 miRNA 靶 mRNA 的一个报告基因。自然发生的 miRNA 海绵被发现是内在表达的植物和动物细胞作为长期的非编码 rna (lncRNAs)17。在本研究中, 我们利用海绵的方法来抑制整个 miR-181 家庭的心脏。虽然 miR-181-sponge 方法是一种生理上相关的方法 downregulate miR-181 家庭在培养细胞,在体内应用 miR-181-sponges 可能是有害的。例如, 一些研究表明, miR-181a 和 miR-181b 在不同细胞/组织类型18,19,20的保护作用。因此, miR-181-sponge 表达应专门针对心脏组织。
通过退火到成熟的 miRNA 导链并防止对 RNA 诱导的沉默复合体 (RISC) 进行适当的加载, 证明小寡核苷酸能阻断 miRNA 功能。与此方法相关的一个问题是, 寡核苷酸必须以饱和剂量提供, 足以阻止成熟 miRNAs 的细胞池。寡核苷酸也面临的挑战涉及跨细胞膜运输和普遍稳定的分子。巧妙地, 它已经表明, 外部提供的 miRNA 目标可以作为一个诱饵, 其同源 miRNA21。由于多个结合点被拴在一起成伪 3 ' UTR 构造, 诱饵目标, 或 miRNA 海绵, 能够稳定地与其目标 miRNAs, 并封存 miRNA microribonucleoprotein 复合体 (miRNPs), 从而有效地防止 miRNA 函数。所谓的 "miRNA 海绵" 是一种有效的抗感技术, 能有效地 downregulate miRNA的体外和体内。因此, miRNA 海绵的应用可以用来研究 miRNA 功能损失表型。
RNA 干扰可能导致一种新的疗法的概念在它的发现以后很快引起了许多调查员的注意。应用的 rna 干扰疗法的领域从实验室迅速地移动了到床边。目前, miRNA 疗法是肿瘤学快速发展的治疗干预之一, 目前正在进行1期临床试验。反义寡核苷酸, 或 antagomirs, 是最广泛使用的 miRNA 抑制方法之一。马等。22使用 miR-10b antagomir, 无论是在体外和体内, 以沉默上调 miR-10b 在一个坚实的肿瘤。
在体内, 有效的沉默上调 miRNAs 需要化学修饰的 antagomirs, 以改善结合亲和性, biostability 和药代动力学的性质。提高双相熔融温度 (Tm), 提高 antagomirs 的核酸酶电阻, 可以进行化学修饰, 如-2′-甲基 (-2′-)、-2′-乙 (-2′--摩尔)-2′-氟和笼锁定核酸 (低噪声放大)23,24,25,26,27,28,29,30,31。为了提高体内分娩的效率, antagomir 可以通过硫代核苷酸 (PS) 的联系进行修改, 从而增加了核酸酶电阻28。此外, PS 骨干的修改也增强了对血浆蛋白的约束, 减少了尿液排泄。因此, PS 修饰 antagomirs 显示了药物动力学性质的显著改善, 促进其系统性分娩32。它还表明, 使用肽核酸 (肽) 或吗啉代寡聚物, 旨在针对特定的 miRNA, 可用于研究 miRNA 功能丧失的表型33,34,35,36,37,38,39. 多聚赖氨酸共轭和纳米颗粒复合肽 antagomirs 有效抑制 miRNA 功能的体外和体内36,37,38,39. 尽管最近取得了进展, 但有效和组织特定地提供 miRNA 衍生物仍然是一项挑战。这些包括潜在的非靶向效应, 触发先天免疫应答, 最重要的是, 获得特定的交付到靶器官/组织的细胞。
另外, 根据细胞和组织类型40, miRNA 表达水平也有很大差异。此外, miRNA 的表达可以增加或减少疾病。miRNA 表达改变的后果和对细胞和组织功能的影响与海绵抑制 miRNA 表达需要得到验证和确定的经验。需要对动物疾病模型进行广泛的临床前研究, 以确定给定 miRNA 靶的最佳抑制水平。
有趣的是, miR-181c miRNA 展览亚细胞划分3,4,5。具体来说, 正如预期的那样, miR-181s 被编码在细胞核中, 转录为长 miRNA 转录, 由划片机在细胞质中处理并纳入 RISC。然而, 与细胞质中作用的典型 miRNAs 不同, miR-181c 的成熟形态 translocates 进入线粒体, 并在调节线粒体特异基因3中发挥作用。结果表明, miR-181-sponge 能将 miR-181c 的成熟形态与细胞质分数结合, 从而防止线粒体的易位。因此, 在 miR-181c 表达下调的条件下, 线粒体中可以观察到 mt-COX1 的上调。
我们报告了设计 miRNA 海绵的方法, 以击倒任何 miRNA 的表达, 并概述了在体内应用 miRNA 海绵的协议。组织特异性 miRNA 抑制是目前 miRNA 领域中的一种技术。下调或功能性抑制上调 miRNAs 在人类疾病中的重要性表明, 有可能利用 miRNA 生物学进行治疗干预。
这项研究强调了一种降低 miRNA 的策略, 它可以在体内以组织特异的方式成功地使用。miRNA 海绵利用 miRNA "种子" 序列的反义序列。因此, miRNA 海绵可以 downregulate 所有共享相同 "种子" 序列的 miRNAs,即整个 miRNA 家庭。在本研究中, 我们观察到了整个 miR-181 家族 (miR-181a、miR-181b、miR-181c 和 miR-181d)在体外和体内表达 miR-181-sponge 的显著下调。如果一个家庭成员给予保护, 而另一个家庭成员在同一器官中有有害的影响, 使用 miRNA 海绵技术将不是理想的方法。

在过去的两年里, 已经有许多研究指出 miRNAs 在人类疾病中的重要作用。大量文献表明, miRNAs 在疾病的病理生理学中具有不可否认的重要性, 如癌症1和心血管疾病2、3、4、5。例如, miR-21 是上调在许多癌症, 导致细胞周期增加和细胞增殖6。在丙型肝炎感染中, miR-122 在病毒7的复制中起着重要作用, 并表明 miR-122 抑制病毒负荷8。在心肌肥厚, miR-212/132 是上调在心脏和参与病理表型9。上调 miRNA 的下调或功能性抑制的明显重要性表明, 在几乎所有疾病中, 治疗利用 miRNA 生物学的机会。
在人类基因组的三个基因组中发现了四 miR-181 家庭成员, miR-181a/b/c/d。非编码 RNA 宿主基因 (MIR181A1-HG) 的内含子区域编码 miR-181-a/b-1 簇。NR6A1 基因的内含子区域对 miR-181-a/b-2 进行编码。miR-181-c/d 星团位于 uncharacterized 19 号染色体上。所有的 miR-181 家庭成员共有相同的 "种子" 序列, 所有四 miR-181 家庭成员可以有可能调节相同的 mRNA 目标。
我们3,4和其他10突出了 miR-181 家庭成员在末期心力衰竭的重要性。我们也认识到, miR-181c 上调发生在与心脏病风险增加有关的病理条件下, 如 II 型糖尿病、肥胖症和衰老3、4、5。据推测, miR-181c 的过度表达会导致氧化应激, 导致心脏功能障碍4。
有几个小组建议 miRNA 存在于线粒体11、12、13、14, 但我们是第一个证明 miR-181c 来源于核基因组、加工和随后移位到线粒体在 RISC3。此外, 我们检测到 miR-181a 和 miR-181b 的低表达在心脏线粒体室5。重要的是, 我们发现 miR-181c 压抑 mt-COX1 mRNA 表达, 从而证明 miRNAs 参与线粒体基因调控和改变线粒体功能3,4。
本文讨论了设计一个 miRNA 海绵, 以摧毁整个 miR-181 家庭在心肌细胞所需的方法。此外, 我们概述了在体内应用的 miR-181-sponge 的协议。

<table><tbody><tr><td>pEGFP-C1 vector</td><td>Addgene</td><td>6084-1</td><td></td></tr><tr><td>In-fusion</td><td>Clontech</td><td>121416</td><td></td></tr><tr><td>QIAprep Miniprep</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>miR-181-sponge synthesis</td><td>Introgen GeneArt</td><td>custome made</td><td></td></tr><tr><td>PCR primers</td><td>Integrated DNA Technologies</td><td>custome</td><td></td></tr><tr><td>EcoRI enzymes</td><td>New Endland Biolabs</td><td>R0101S</td><td></td></tr><tr><td>KpnI enzymes</td><td>New Endland Biolabs</td><td>R0142S</td><td></td></tr><tr><td>Rapid DNA Ligation Kit</td><td>Sigma-Aldrich</td><td>11635379001</td><td></td></tr><tr><td>H9c2 cells</td><td>ATCC</td><td>CRL-1446</td><td></td></tr><tr><td>DMEM Media</td><td>Thermo Fisher Scientific</td><td>11965092</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>10082139</td><td></td></tr><tr><td>Nucleofector 2b Device</td><td>Lonza</td><td>AAB-1001</td><td></td></tr><tr><td>Nucleofector Kits for H9c2 (2-1)</td><td>Lonza</td><td>VCA-1005</td><td></td></tr><tr><td>G418, Geneticin</td><td>Thermo Fisher Scientific</td><td>11811023</td><td></td></tr><tr><td>FACSAria II Flow cytometer</td><td>BD Bioscience</td><td>644832</td><td></td></tr><tr><td>Branson 450 sonifier</td><td>Marshall Scientific</td><td>EDP 100-214-239</td><td></td></tr><tr><td>The Xenogen IVIS Spectrum optical imaging device</td><td>Caliper Life Sciences</td><td></td><td></td></tr><tr><td>Anti-MTCO1 antibody</td><td>Abcam</td><td>ab14705</td><td></td></tr><tr><td>&amp;alpha;-tubulin antibody</td><td>Abcam</td><td>ab7291</td><td></td></tr><tr><td>Sequoia C256 ultrasound system</td><td>Siemens</td><td></td><td></td></tr></tbody></table>

in vivo nanovector delivery of a heart-specific microrna-sponge

MicroRNA (miRNA) 是一种小的非编码 rna, 它抑制转录后信使 rna (mRNA) 的表达。人类疾病, 如癌症和心血管疾病, 已被证明激活组织和/或细胞特异的 miRNA 表达与疾病进展有关。miRNA 表达的抑制提供了治疗干预的潜力。然而, 传统的抑制 miRNAs 的方法, 使用 antagomir 寡核苷酸, 影响特定的 miRNA 功能, 在全球交付。本文提出了一种在大鼠模型中对 miR-181 家族进行心脏特异性抑制的协议。miRNA 海绵结构的设计包括10重复 anti-miR-181 绑定序列。心脏特异的α MHC 启动子被克隆成 pEGFP 骨干, 以驱动心脏特异的 miR-181 miRNA 海绵表达。为了创建一个稳定的细胞系表达 miR-181-sponge, 骨骼肌 H9c2 细胞被转染的α-MHC-EGFP 和平号-181 海绵结构, 并排序由荧光活化细胞分类 (资产管制署) 到 GFP 阳性 H9c2 细胞培养与新霉素(G418)。随着新霉素的稳定增长, 单克隆细胞的数量是由额外的和单细胞克隆建立的。由此产生的骨骼肌 H9c2-miR-181-sponge-GFP 细胞表现为 miR-181 家庭成员的功能丧失, 这是通过增加 miR-181 靶蛋白的表达来评估的, 而与表达非功能性海绵的 H9c2 细胞相比。此外, 我们还开发了一个 nanovector 的系统交付 miR-181-sponge 结构的络合正电荷脂质体纳米粒子和负电荷 miR-181-sponge 质粒。在活体成像的 GFP 显示, 多尾静脉注射的 nanovector 在三周的时间, 能够促进一个重要的表达 miR-181-sponge 在心脏特定的方式。重要的是, 在心脏组织, 而不是在肾脏或肝脏中观察到 miR-181 功能的丧失。miRNA 海绵是抑制组织特异 miRNA 表达的有力方法。驱动 miRNA 海绵表达从组织特定的启动器提供特异性的 miRNA 抑制, 这可以被限制在靶器官或组织。此外, 结合 nanovector 和 miRNA 海绵技术, 允许有效的分娩和组织特定的 miRNA 抑制在体内。

在稳定转染的 pEGFP-miR-181-sponge-expressing H9c2 细胞 (从步骤 4.2), 整个 miR-181 家族 (miR-181a, miR-181b, miR-181c 和 miR-181d) 的表达相对于 pEGFP--表达 H9c2 细胞有适度的减少。MiR-181-sponge 作为整个 miR-181 家族的一个竞争性抑制剂, 因此我们预计 miR-181c 线粒体靶基因的表达将会增加, mt-COX1。西方的印迹数据表明, pEGFP-miR-181-sponge-expressing H9c2 细胞中的 mt-COX1 表达比 pEGFP-爬行表达的 H9c2 细胞增加。此外, 在 pEGFP-miR-181-sponge-expressing H9c2 单元格中通过应用免疫印迹检测到 mt-COX2 或 mt-COX3 的表达式没有变化。我们用α蛋白作为免疫印迹法分析的规范化控制。GFP 信号主要观察在肝脏和肾脏长达2周后系统交付的和平号海绵 nanovector (图 1A)。然而, 在3周 miR-181-sponge nanovector 分娩后, GFP 的表达明显高于心脏组织 (图 1A和1B)。注意, 在系统分娩3周后, 某些动物的肝脏组织中检测到了 GFP 信号;然而, miR-181-sponge 在当时的肝脏中没有功能性作用。
miR-181-sponge nanovector 注射大鼠的 mt-COX1 表达明显增加, 与争夺 nanovector 组相比 (图 2)。较高的 mt-COX1 表明, 在 miR-181-sponge nanovector 分娩后的3周内, 心脏 miR-181c 表达较低, mt-COX1 是 miR-181c 的直接目标。我们用α蛋白作为 immunoblots 的规范化控制。
超声心动图显示, miR-181-sponge nanovector 注射大鼠在治疗方案结束前、期间和末期的心功能没有改变, 与 nanovector 群动物的争夺相比。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57845/57845fig1.jpg"> 图 1.体内miR-181-Sponge nanovector 的分析.(A) 这个小组显示了经过优化的3周治疗方案, 6 静脉注射通过尾静脉。荧光图像中的黄色着色演示了 pEGFP 向量的表达式。黄色着色的最大强度在星期3时间点被观察。(B) pEGFP 向量中的α MHC 启动子序列有选择地表示在21天心脏的 miR-181-sponge 或被打乱的序列。<a href="https://www.jove.com/files/ftp_upload/57845/57845fig1large.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/57845/57845fig2.jpg"> 图2。验证 miR-181-sponge nanovector 对 miR-181c 表达的影响.这是西方印迹分析的 mt-COX1 表达在心脏裂解物获得的 pEGFP 和 pEGFP-miR-181-sponge nanovector 注射大鼠。用所述抗体对全心组织匀浆进行了探讨。我们用α蛋白作为装载控制。带密度测量显示在下面的条形图中。对学生进行t检验, 在条形图中绘制标准误差作为误差条。* p < 0.05与pEGFP 集团。n = 4。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>底漆名称</td>
			<td colspan="2">序列</td>
			<td>Tm</td>
			<td>笔记</td>
			<td>使用</td>
			<td>目标</td>
		</tr>
<tr>
<td>SAM3-1F</td>
			<td colspan="2">ATGCATTAGTTATTAATGCTT GACACACTTGACAATTTCT</td>
			<td>58。4</td>
			<td>大鼠 MHC 启动子克隆成 EGFP</td>
			<td>Pcr</td>
			<td>MHC 启动子</td>
		</tr>
<tr>
<td>SAM3-2R</td>
			<td colspan="2">GACCGGTAGCGCTAGCTG ACTCACTGGGAGATTGCTT</td>
			<td>59。8</td>
			<td>大鼠 MHC 启动子克隆成 EGFP</td>
			<td>Pcr</td>
			<td>MHC 启动子</td>
		</tr>
<tr>
<td>SAM3-3F</td>
			<td colspan="2">CGAACGACCTACACCGAACT</td>
			<td>64</td>
			<td>屏幕 EGFP-F</td>
			<td>菌落 PCR</td>
			<td>正启动子克隆</td>
		</tr>
<tr>
<td>SAM3-4R</td>
			<td colspan="2">CGCTAGTCCTTGACCCTCTG</td>
			<td>63。9</td>
			<td>屏幕 MHC R</td>
			<td>菌落 PCR</td>
			<td>正启动子克隆</td>
		</tr>
<tr>
<td>SAM5-1F</td>
			<td colspan="2">CCTGTCTCCAACACACAAGC</td>
			<td>59。3</td>
			<td>序列 MHC 启动子</td>
			<td>测 序</td>
			<td>MHC 启动子</td>
		</tr>
<tr>
<td>SAM5-2R</td>
			<td colspan="2">CAGACTGCAGGGCTGGTT</td>
			<td>60</td>
			<td>序列 MHC 启动子</td>
			<td>测 序</td>
			<td>MHC 启动子</td>
		</tr>
</tbody></table>表1。底漆序列。

Watch this Scientific Journal Video about In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge at JoVE.com

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

体内Nanovector 心脏特异性 MicroRNA 海绵的交付

组织特异性 microRNA 抑制是一种在 microRNA 领域发展不发达的技术。在这里, 我们描述了一个协议, 以成功地抑制 miR-181 microRNA 家族在骨骼肌细胞从心脏。Nanovector 技术是用来提供一个 microRNA 海绵, 表明重要的体内心脏特定 miR-181 家庭抑制。

MicroRNA (miRNA) is small non-coding RNA which inhibits post-transcriptional messenger RNA (mRNA) expression. Human diseases, such as cancer and ...

in-vivo-nanovector-delivery-of-a-heart-specific-microrna-sponge

Nanyang Technological University

Research

JoVE Journal

Biology

In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

7.3K 次观看.  University of Toronto. 组织特异性 microRNA 抑制是一种在 microRNA 领域发展不发达的技术。在这里, 我们描述了一个协议, 以成功地抑制 miR-181 microRNA 家族在骨骼肌细胞从心脏。Nanovector 技术是用来提供一个 microRNA 海绵, 表明重要的体内心脏特定 miR-181 家庭抑制。

视频: In Vivo Nanovector Delivery of a Heart-specific MicroRNA-sponge

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 ...

科研

遗传学

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

纳米粒子介导的 siRNA 基因沉默在斑马鱼心脏中的表达

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

发育生物学

<meta charset="utf8"></head><body>靶向致癌 miRNA-10b 治疗转移性乳腺癌的纳米药物

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or &#34;nanodrug&#34;) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

<meta charset="utf8"></head><body>监测小鼠乳腺癌转移灶中纳米药物的积累

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

监测小鼠乳腺癌转移灶中纳米药物的积累

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have ...

癌症研究

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

一个简单的替代，以立体定向注射miRNA的脑特异性敲除

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

神经科学

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

制备和<em&gt;体外</em&gt;磁化的表征的miR-改性内皮细胞

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

医学

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

长期沉默的1S Intersectin的在小鼠肺的特异性siRNA，通过阳离子脂质体重复交货。通过电子显微镜的击倒效果评价

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

生物学

<meta charset="utf8"></head><body>用于体内基因调节的慢病毒载体制备

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue&#45;Resident Macrophages In Vivo in Mice

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

<meta charset="utf8"></head><body>作者聚焦:通过慢病毒载体介导的基因操作探索巨噬细胞免疫代谢

We demonstrate a step-by-step protocol for the investigation of gene function in peritoneal tissue-resident macrophages in vivo, using lentiviral vectors.

小鼠 体内 腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring ...

体内Nanovector 心脏特异性 MicroRNA 海绵的交付

摘要

探索更多视频

此视频中的章节

在心肌梗死小鼠模型中传递修正的mRNA

纳米粒子介导的 siRNA 基因沉默在斑马鱼心脏中的表达

监测小鼠乳腺癌转移灶中纳米药物的积累

监测小鼠乳腺癌转移灶中纳米药物的积累

监测小鼠乳腺癌转移灶中纳米药物的积累

一个简单的替代，以立体定向注射miRNA的脑特异性敲除

制备和

长期沉默的1S Intersectin的在小鼠肺的特异性siRNA，通过阳离子脂质体重复交货。通过电子显微镜的击倒效果评价

小鼠体内腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备

小鼠体内腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备

体内Nanovector 心脏特异性 MicroRNA 海绵的交付

摘要

探索更多视频

此视频中的章节

在心肌梗死小鼠模型中传递修正的mRNA

纳米粒子介导的 siRNA 基因沉默在斑马鱼心脏中的表达

监测小鼠乳腺癌转移灶中纳米药物的积累

监测小鼠乳腺癌转移灶中纳米药物的积累

监测小鼠乳腺癌转移灶中纳米药物的积累

一个简单的替代，以立体定向注射miRNA的脑特异性敲除

制备和

长期沉默的1S Intersectin的在小鼠肺的特异性siRNA，通过阳离子脂质体重复交货。通过电子显微镜的击倒效果评价

小鼠 体内 腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备

小鼠 体内 腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备

小鼠体内腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备

小鼠体内腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备