Name: 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
Uploaded: 2013-06-21T13:00:00
Description: Watch this Scientific Journal Video about Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Elect

支持这项工作是由国家卫生赠款R01HL089462 SP研究所。

1。阳离子脂质体的制备<ol><li>高压灭菌用于lipososmes制备一个干净的圆底烧瓶中，。 </li><li>准备股票的解决方案： <ol class="lalpha"><li> - 在10毫升氯仿中，使用一个小的玻璃瓶中溶解200毫克的二甲基二（十八烷基）溴化铵（DOAB）。 </li><li> - 200毫克的胆固醇溶解在10毫升氯仿，用一个小玻璃瓶。 </li><li> - 准备和高压灭菌器进行灭菌的5％葡萄糖，在100毫升的RNA / DNA酶的蒸馏水。 </li></ol></li><li>冲洗圆...

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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ-specific+endothelial+cell+uptake+of+cationic+liposome-DNA+complexes+in+mice.">Organ-specific endothelial cell uptake of cationic liposome-DNA complexes in mice.</a> Am. J. Physiol. (273), H387-H404 (1997).</li><li>Soutschek, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+silencing+of+an+endogenous+gene+by+systemic+administration+of+modified+siRNAs.">Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs.</a> Nature. (432), 173-178 (2004).</li><li>Barenholz, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes+enhance+bioremediation+of+oil-contaminated+soil.">Liposomes enhance bioremediation of oil-contaminated soil.</a> J. Liposome Res. (13), 1-8 (2003).</li><li>Zamboni, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal,+nanoparticle,+and+conjugated+formulations+of+anticancer+agents.">Liposomal, nanoparticle, and conjugated formulations of anticancer agents.</a> Clin. Cancer Res. (11), 8230-8234 (2005).</li><li>Newman, M. S., Colbern, G. T., Working, P. K., Engbers, C., Amantea, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+pharmacokinetics,+tissue+distribution,+and+therapeutic+effectiveness+of+cisplatin+encapsulated+in+long-circulating,+pegylated+liposomes+(SPI-077)+in+tumor-bearing+mice.">Comparative pharmacokinetics, tissue distribution, and therapeutic effectiveness of cisplatin encapsulated in long-circulating, pegylated liposomes (SPI-077) in tumor-bearing mice.</a> Cancer Chemother Pharmacol. (43), 1-7 (1999).</li><li>Martino, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+siRNA+delivery+by+the+cationic+liposome+DOTAP+in+human+hematopoietic+stem+cells+differentiating+into+dendritic+cells.">Efficient siRNA delivery by the cationic liposome DOTAP in human hematopoietic stem cells differentiating into dendritic cells.</a> J. Biomed. Biotechnol. (2009), 410260 (2009).</li><li>Zhang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+delivery+system+targeting+bone+formation+surfaces+to+facilitate+RNAi-based+anabolic+therapy.">A delivery system targeting bone formation surfaces to facilitate RNAi-based anabolic therapy.</a> Nat. Med. 18 (18), 307-314 (2012).</li><li>Predescu, D., Palade, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmalemmal+vesicles+represent+the+large+pore+system+of+continuous+microvascular+endothelium.">Plasmalemmal vesicles represent the large pore system of continuous microvascular endothelium.</a> Am. J. Physiol. (265), H725-H733 (1993).</li><li>Koh, T. W., Verstreken, P., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dap160/intersectin+acts+as+a+stabilizing+scaffold+required+for+synaptic+development+and+vesicle+endocytosis.">Dap160/intersectin acts as a stabilizing scaffold required for synaptic development and vesicle endocytosis.</a> Neuron. (43), 193-205 (2004).</li><li>Miyawaki-Shimizu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lung Cell Mol. Physiol. (290), L405-L413 (2006).</li></ol>

根据以往的研究公布的其他9和我们1，18，我们开发的这种方法长期打倒的ITSN-1 在体内特异性siRNA /脂质体复合物反复静脉给药（每72小时，连续24天）。这种实验的方法是有效率的，可以安全地使用和重复，它可以容易地扩展到研究肺血管内皮细胞和肺稳态感兴趣的任何蛋白质的编码基因的参与。我们成立了由重复的siRNA /脂质体交付的实验条件下，小鼠出现毒性或免疫反应?...

裸siRNA的不能穿透细胞膜，被带负电荷的，它很容易降解酶在血液，组织和细胞。即使最近的结构修改，以提高稳定性，在目标站点的siRNA积累给药后非常低，需要一个高效的细胞内车辆5。阳离子脂质体成为安全核酸载体的电位转移到细胞中的DNA / RNA的大块的封装和保护核酸酶降解6。此外，自发的交互阳离子脂质体与DNA / RNA，从而促进基因转移到细胞2。最近的脂质体已被应用到提供疫苗和低分子量药物7。脂质体交付的microRNA-7表达质粒克服表皮生长因子受体酪氨酸激酶抑制剂的耐药性在肺癌细胞中8。当目标血管内皮细胞，静脉内发送的配合物是必不可少的，因为不可能穿过内皮屏障和外渗到间质9。到其他器官相比，从肺微血管内皮细胞有最大的吸收，贪婪地内在化的阳离子脂质体与DNA / RNA复合物，其次是淋巴结和Peyer氏斑9。静脉交付的siRNA通过后眼窝或尾静脉注射的啮齿动物模型中已证明是无害的，即使在高浓度，如50毫克/公斤10。在公开发表的文献中，在阳离子脂质体组合物，不同的基础上的脂质制剂和它们的等摩尔比11，12。有广泛的潜在的应用，使用阳离子脂质体在体内的基因传递，无论目标的蛋白质down-regulation/over-expression，交付的疫苗或抗肿瘤治疗13-15。重要的是记住是，DNA / RNA的细胞的膜相互作用的效率之间的耦合产生的复合物和膜脂质的形状受。这表明，定制的目标细胞膜公司的脂质组合物，可能是有益的，并导致在一个特定的细胞类型6的高转染率。

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimethyl Dioctadecyl Ammonium Bromide (DOAB)</td>
 <td>Sigma-Aldrich</td>
 <td>D2779</td>
 <td></td>
 </tr>
 <tr>
 <td>LiposoFast stabilizer (mini-extruder)</td>
 <td>Avestin Inc.</td>
 <td>LF-STB</td>
 <td></td>
 </tr>
 <tr>
 <td>Polycarbonate membrane, 19 mm diameter, 50 nm pore</td>
 <td>Avestin Inc.</td>
 <td>LFM-50</td>
 <td></td>
 </tr>
 <tr>
 <td>Cholesterol</td>
 <td>Sigma-Aldrich</td>
 <td>C-8667</td>
 <td></td>
 </tr>
 <tr>
 <td>Chloroform</td>
 <td>Mallinckrodt Chemicals</td>
 <td>4432-04</td>
 <td></td>
 </tr>
 <tr>
 <td>Hank's balanced salts</td>
 <td>Sigma-Aldrich</td>
 <td>H1387</td>
 <td></td>
 </tr>
 <tr>
 <td>Round-bottom flask</td>
 <td>Fisher Scientific</td>
 <td>FHB-275-030X</td>
 <td></td>
 </tr>
 <tr>
 <td>Mouse siRNAITSN - on target </td>
 <td>Dharmacon/ThermoScientific</td>
 <td>J-046912-11</td>
 <td></td>
 </tr>
 <tr>
 <td>Peristaltic pump</td>
 <td>Ismatec</td>
 <td>REGLO-CDF digital with RHOO pump head</td>
 <td></td>
 </tr>
 <tr>
 <td>Ventilator</td>
 <td>Hugo Sachs Electronik</td>
 <td>NA</td>
 <td></td>
 </tr>
 <tr>
 <td>Barbital</td>
 <td>Sigma-Aldrich</td>
 <td>B-0500</td>
 <td></td>
 </tr>
 <tr>
 <td>Uranyl acetate</td>
 <td>EM Sciences</td>
 <td>22400</td>
 <td></td>
 </tr>
 <tr>
 <td>Epon812</td>
 <td>EM Sciences</td>
 <td>Discontinued by the manufacturer</td>
 <td></td>
 </tr>
 <tr>
 <td>Propylene oxide</td>
 <td>EM Sciences</td>
 <td>20400</td>
 <td></td>
 </tr>
 <tr>
 <td>Embedding molds</td>
 <td>EM Sciences</td>
 <td>69923-05</td>
 <td></td>
 </tr>
 <tr>
 <td>Tannic acid</td>
 <td>EM Sciences</td>
 <td>21710</td>
 <td></td>
 </tr>
 <tr>
 <td>8 nm gold-albumin tracer</td>
 <td></td>
 <td>Prepared in the laboratory as in16</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Comments</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipped with 2 Avestin 1 ml gas-tight syringes (cat. # LF-1)</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Pyrex, 100 ml</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Modified siRNA, in vivo</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Part of IDEX corp.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>D-79232 March, Germany</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Replaced with Embed812, cat. # 14120</td>
 </tr>
 </tbody>
</table>

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

以往的研究表明，击倒的ITSN-1S（KD ITSN），内吞蛋白参与调节肺血管通透性和内皮细胞（ECS）的生存，诱导细胞凋亡，长时间ITSN-1的细胞培养系统开发的主要障碍抑制1。使用阳离子脂质体为载体，我们探讨了沉默的ITSN-1基因在小鼠肺组织全身的的siRNA靶向ITSN-1基因（的siRNA ITSN）的管理。阳离子脂质体siRNA传递提供了几个优点：重复给药的安全，无免疫原性，无毒，易产生2。脂质体的性能和生物活性取决于其大小，电荷，脂质成分，稳定，剂量和路由管理3在这里，效率和具体的KD ITSN在小鼠肺部已获得胆固醇和二甲基二十八烷基三甲基溴化铵的组合。静脉交付的的siRNA ITSN /阳离子脂质体复合物瞬间撞倒-1蛋白和mRNA ITSN在小鼠肺部的第3天，其中痊愈后3天。以利用作为一个可重复的安全载体的阳离子脂质体，研究的24天延长。因此，眼窝治疗刚生成的络合物每第三天，诱导持续KD ITSN的整个研究4。将进行小鼠组织收集的几个时间点后的siRNA ITSN的电子显微镜（EM）分析，以评估影响慢性KD ITSN，肺血管内皮细胞中。高分辨率EM成像使我们能够评估在肺血管床（ 即破坏内皮屏障由KDITSN引起的形态变化，数量减少的小窝和其他交通途径的上调），特点非光显微镜检测。总体而言，这些研究结果建立IMPOR桑特作用ITSN-1在内皮细胞功能和肺稳态，同时说明在体内的siRNA脂质体输送的有效性。

在几个时间点后的siRNA ITSN的交付Western blot和常规和定量PCR 4-1S ITSN的蛋白和mRNA水平进行了监测。在处理的siRNA ITSN的小鼠肺组织的-1S ITSN的蛋白和mRNA水平低约75％控制在21天连续击倒ITSN-1S。如果没有-1 ITSN，动力2，一个主要的相互作用合作伙伴的1s和ITSN必不可少播放器脱离从质膜的小窝还没有有效的招募的内吞站点，从而小窝脱离质膜和形成自由水泡性口载流子被打乱1?...

Watch this Scientific Journal Video about Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Elect

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 (Video) | JoVE

重复眼窝的阳离子liposomes/siRNA/ITSN-1s的复合物在小鼠24天，每72小时注射，有效地传递siRNA双链由75％减少-1S ITSN的mR​​NA和蛋白表达的小鼠肺微血管。这种技术是高度重现性的动物模型中，没有不利影响，并避免了死亡。

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 ...

Research

JoVE Journal

Biology

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

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

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长期使用宽视场显微镜的成像哺乳动物细胞

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