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>冲洗圆底烧瓶中，用氯仿。添加5-10毫升的烧瓶中，漩涡，并让它坐几分钟。用铝箔纸覆盖在任何时候都保持烧瓶。清空从烧瓶中的氯仿。 </li><li>脂质体的制备: <ol class="lalpha"><li> - 添加315微升的DOAB原液圆底烧瓶。 </li><li> - 胆固醇原液向烧瓶中加入200微升。 </li><li> - 添加9.5毫升氯仿烧瓶第二漩涡混合。 </li></ol></li><li>设置旋转蒸发仪在37℃，100每分钟转速（RPM）或更多（100-120转）。应连接到旋转蒸发器与吸入管连接正确的水泵的真空控制。 </li><li>连接氩气槽和旋转蒸发仪。 </li><li>将烧瓶旋转蒸发仪，并降低在水浴前调整最佳设置。氩气完全打开第一阀逆时针转动。务必打开和关闭该阀。第二阀控制吹在向烧瓶中的氩气压力。压力应该总是温柔。 </li><li>在水中洗澡，足以放下旋转蒸发烧瓶碰水没有被完全淹没，和打开的速度。 </li><li>等待，直到所有的氯仿蒸发，约10分钟。然后停止机器。 </li><li>旋转蒸发速度和氩罐阀关闭。载入该烧瓶中应被删除。 </li><li>加入2毫升的5％葡萄糖日ê烧瓶中溶解血脂。 </li><li>刮开使用1毫升枪头彻底溶解血脂的烧瓶底部。划伤后，除去任何剩余的脂质从1毫升尖端到烧瓶中，如果必要的话，与第二前端。 </li><li>一边慢慢涡旋（每分钟15转）的烧瓶中，将得到的溶液在42℃水浴中孵育。 </li><li>填写超声波破碎大桶冷水。超声溶液与举起的烧瓶中，并刚好接触水的底部至少20-30分钟。 </li><li>的烧瓶中，在旋转蒸发器中的水浴加热20分钟，在42℃下，在零速时，与浸在水中，这时候的圆底。 </li><li>脂质体通过0.47微米，随后，0.22微米的针头式过滤器过滤。 </li><li>使用一个小的挤出机中，过滤通过50 nm的膜脂质体的单室脂​​质体囊，以产生均匀的人口。 </li><li>脂质体中转移至Eppendorf管中，并置于冰上。 </li></ol><html> 2。准备脂质体:的siRNA ITSN的配合<ol><li>重悬上的目标的小鼠的siRNA ITSN的 siRNA中含有20mM HEPES和150mm的氯化钠，pH值7.4的缓冲液至终浓度为2微克/微升。混合之前保持在冰上。 </li><li> 8 nmol脂质体:2微克RNA（400 nmol脂质体100微克的siRNA ITSN的 ），上面的比例制备的脂质体:的siRNA ITSN络合物。在本研究中，我们从储备溶液（50μM）的siRNA ITSN的 50微升新鲜制备的脂质体中使用的RNA / DNA的酶的Eppendorf管中，加入100μl。注意浓度的siRNA ITSN是非常重要的，因为最大为150微升的混合物，可以在同一时间在小鼠双边和眶注入。 </li><li>等待注入小鼠的同时，保持该混合物在冰上。 </li></ol> 3。鼠标眼窝静脉注射（内部角度眼圈） jove_content"&gt;所有小鼠实验进行了批准和执行根据拉什大学机构动物护理和使用委员会的指导方针。 <ol><li>麻醉小鼠腹腔注射50毫克/公斤体重的氯胺酮和5毫克/公斤体重甲苯噻嗪（市售的混合物）。根据动物的体重和年龄的不同，调整的麻醉剂的量和浓度。所用小鼠在研究小鼠CD1雄性小鼠6-8周龄，体重约25克。结核菌素注射器1毫升型针头。 </li><li>用一只手按住鼠标耳朵，并把鼠标的一侧的眼睛，露出内部的角度。瞄准内侧皱襞半月;避免接触眼球。 </li><li>接近持注射器含有混合物在所有三个轴45度角的眼睛泪阜。 </li><li>缓缓注入混合物让鼠标恢复注入侧。如果有大量液体液滴形式针头插入的地方，退针，倒吸液滴入注射器，然后再试一次。反复注射是最好由交替的眼睛来完成，让完美的复苏。如果不确定，这种技术可以检查，通过注入蓝色染料（50微升，甲醇0.5％龙胆紫）和暴露小鼠肺组织观察蓝色血管。 </li><li>在这项研究中的小鼠注射每3周的第 3天，无死亡或不良影响。 </li></ol> 4。生化研究小鼠肺灌注和组织收集<ol start="0"><li>设置在蠕动泵运行在1.5毫升/分钟。 </li><li>准备设置:呼吸机，手术台，器械，无菌棉签，棉签，字符串，烧杯，蒸馏水，汉克的平衡盐，示踪。 </li><li>麻醉小鼠腹腔注射麻醉剂的混合物（100毫克/公斤体重的氯胺酮和10毫克/公斤体重甲苯噻嗪）。 </lI&gt; <li>的颈部，取出唾液腺暴露气管。 </li><li>开始剖腹探查，切除隔膜，并按照与开胸，暴露肺和心脏的。 </li><li>取出胸腺。 </li><li>更准确地使用光镜，导尿肺动脉。 </li><li>做气管切开及插管鼠标采用气管造口。将呼吸机的速度150次/分，每搏输出量150微升。 </li><li>作为插座，打开左心房。 </li><li>灌注的小鼠肺脉管血液5分钟，使用蠕动泵和Hank溶液在37℃下加热</li><li>灌注示踪剂（8 nm金白蛋白16）额外的10分钟，在相同的流速。 </li><li>冲洗未结合的示踪肺灌注5分钟，汉克的解决方案。 </li><li>关注的肺部原位固定10分钟的灌注4％（重量/体积）的甲醛，2.5％戊二醛，1％鞣酸0.1筒缓冲液，pH 7.2。 </li><li>收集肺，除去多余的组织，切小块（3/3毫米），并把它们标记闪烁小瓶含2毫升混合固定液。 </li></ol>老鼠会在手术过程中麻醉完全失效。 5。小鼠肺组织处理EM <ol><li>准备股票解决方案:1％醋酸巴比妥，帕拉德OSO 4，克伦贝格尔缓冲区。 <ol class="lalpha"><li> - 1％帕拉德-OSO 4缓冲液包含:1毫升醋酸巴比妥的库存，1.25毫升4％OSO 4，1毫升的0.1当量的盐酸，蒸馏水5毫升的最终体积。 </li><li> - 醋酸巴比妥库存溶液溶解1.15的乙酸钠andydrous和2.94克巴比妥在100毫升蒸馏水中通过以下方式获得。 </li><li>克伦贝格尔溶液包含2毫升醋酸巴比妥库存，2.8毫升0.1N盐酸，醋酸双氧铀0.05克，和5.1毫升的蒸馏水，pH值= 6。 </li></ol></li><li>将猪肺洗净块中的0.1M的二甲胂酸钠缓冲液，pH = 7.4的，短暂的3倍。 </li><li>固定在试样中的4％（重量/体积）的甲醛，2.5％戊二醛在0.1 PIPES缓冲液，pH值7.2，1小时后，在室温下。 </li><li>修复后用1％锇帕拉德在引擎盖1小时，在冰上，在黑暗中。 </li><li>冲洗一次与克伦贝格尔缓冲区。 </li><li> 2小时至过夜，在室温下孵育克伦贝格尔缓冲液标本。 </li><li>在50％乙醇中冲洗一次。 </li><li>梯度乙醇，5分钟/每:70％，95％，则2×15分钟100％乙醇脱水。 </li><li>切换到100％的环氧丙烷，2×15分钟。 </li><li>取出环氧丙烷的混合物加入50％的环氧丙烷和50％EPON 812，孵育过夜，旋转的车轮上，在室温下。 </li><li>第二天早上，取出该混合物，并加入新鲜的EPON 812，至少在4-5小时在车轮上。 </li><li>使用一倍锥形模具，填充半路上，新鲜的100％EPON 812，添加选定的标本，并把它们的边缘。 </li><li>将模具在60℃的孵化器集，并让他们固化48-72小时。 </li><li>聚合时，发送块要薄（60-70纳米厚）切片。 </li></ol>

<ol>
	<li>Predescu, S. A., Predescu, D. N., Knezevic, I., Klein, I. K., Malik, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intersectin-1s+regulates+the+mitochondrial+apoptotic+pathway+in+endothelial+cells.">Intersectin-1s regulates the mitochondrial apoptotic pathway in endothelial cells.</a> J. Biol. Chem. 282 (282), 17166-17178 (2007).</li><li>Uddin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+lipids+used+in+non-viral+gene+delivery+systems.">Cationic lipids used in non-viral gene delivery systems.</a> Biotechnology and Molecular Biology Review. 2 (2), 58-67 (2007).</li><li>Barenholz, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+lipid+composition+on+the+thermotropic+behavior+and+size+distribution+of+mixed+cationic+liposomes.">Influence of lipid composition on the thermotropic behavior and size distribution of mixed cationic liposomes.</a> J. Colloid Interface Sci. (356), 46-53 (2011).</li><li>Predescu, D. N., Neamu, R., Bardita, C., Wang, M., Predescu, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+caveolae+function+and+upregulation+of+alternative+endocytic+pathways+induced+by+experimental+modulation+of+intersectin-1s+expression+in+mouse+lung+endothelium.">Impaired caveolae function and upregulation of alternative endocytic pathways induced by experimental modulation of intersectin-1s expression in mouse lung endothelium.</a> Biochem. Res. Int.. (2012), 672705 (2012).</li><li>Higuchi, Y., Kawakami, S., Hashida, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+in+vivo+delivery+of+siRNAs:+recent+progress.">Strategies for in vivo delivery of siRNAs: recent progress.</a> BioDrugs. 24 (24), 195-205 (2010).</li><li>Caracciolo, G., Amenitsch, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+liposome/DNA+complexes:+from+structure+to+interactions+with+cellular+membranes.">Cationic liposome/DNA complexes: from structure to interactions with cellular membranes.</a> Eur. Biophys. J. , (2012).</li><li>Barnier Quer, C., Elsharkawy, A., Romeijn, S., Kros, A., Jiskoot, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+liposomes+as+adjuvants+for+influenza+hemagglutinin:+more+than+charge+alone.">Cationic liposomes as adjuvants for influenza hemagglutinin: more than charge alone.</a> Eur. J. Pharm. Biopharm. 81 (81), 294-302 (2012).</li><li>Rai, 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=Liposomal+delivery+of+MicroRNA-7-expressing+plasmid+overcomes+epidermal+growth+factor+receptor+tyrosine+kinase+inhibitor-resistance+in+lung+cancer+cells.">Liposomal delivery of MicroRNA-7-expressing plasmid overcomes epidermal growth factor receptor tyrosine kinase inhibitor-resistance in lung cancer cells.</a> Mol. Cancer Ther. (10), 1720-1727 (2011).</li><li>McLean, J. 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>

什么都没有透露。

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

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 ...

long-term-silencing-intersectin-1s-mouse-lungs-repeated-delivery

Research

JoVE Journal

Biology

10.6K 次观看.  Rush University. 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。

视频: 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

Actually what I'm gonna do, mount it in here first and then swap out You. So now I'm gonna aspirate try and be as gentle as possible.Okay. A few seconds just holding.Okay.Alright.So the important features of our lifestyle imaging grid are that, so it's an inverted scope, which means the objective, the samples here in the objective is underneath. So the objective is pointing up and the whole scope, or practically the whole, practically the whole scope, including the objectives and the sample are at 37 degrees. So everything in this plastic box is at 37 degrees.And right on top of the stage we have an air and CO2 mix. That is 5%CO2. So the idea is that the sample is actually in a cell, it's like a cell incubator.And so we have, this is our mixer for the air and the CO2 and, and it gets humidified and heated and then piped onto the, the sample on the stage.Okay. And today we're just gonna be doing fluorescence one channel GFP fluorescence. This is the hose that the CO2 and 5%CO2 goes in and it just fits into the stage holder.That's important. And then we can close these, take these down, and then we go to our first position. And what I'm gonna do is go to the first field, which is my control sample, and I'll pick positions based on what I see on the screen.So I just make sure I actually look into the microscope in each new, well, because the focal plane for each of the wells is different, the, the dish is not perfectly flat on the bottom. So you will need to make fine adjustments when you move from well to the well. So right now I'm on the first, well, what I'll do is I'll focus, so I just hit, I just told, I just turned on the light, the transmitted light for the microscope.And I have make sure all the light is going to the eye pieces. And so I just look in, make sure I get a nice clean field. And then what I'll do is turn that light off, turn the fluorescence light on, make sure the cells that I'm looking at look healthy and they're not apoptotic.And, and I turn that off. And what I do next is send, so now I'm gonna start picking positions, which means I have to te send the light path to the camera. This is the camera right here.So I tell the microscope to send all the camera, they don't see anything in the eye pieces. What I'll do is in the software controlling the microscope, I'll choose the right filter set, which for our purposes is psy. And then set an exposure time.And I know these cells very well and I know that they take a 0.2 second exposure time. So I tell, that's what I tell the software to do. And what I'll do is hit focus.This is, and it's basically just alive. The, A light will turn on and you can, oh, I don't know if you can see cells, but the reason this is a good field is because there's, all the cells are, even in terms of their fluorescence, There are no apoptotic cells. Apoptotic cells tend to be very bright and there's no dirt or schmutz in the field.And you can see that they're actually, this is a metaphase cell and that's a metaphase cell, that's a cell that's probably just exited mitosis. And I can just tell that because it looks like those are, those two sets of chromatin are very nicely oriented to each other. Like they just exited an face.So that's a good field. So, and now that I know that the exposure time and the filter set is correct, and this is gonna be software specific, I'll go and I'll take positions and now the software has memorized the X, Y, Z position of that field. And then when I do pick another field, it's, and I'll, so when I'm picking, when I'm picking new fields, but in the same, well, I'll use the, I'll move the i'll, I'll turn the shutter, I'll open the shutter and I'll look to see what I see on the screen and I'll pick fields based on that versus looking in the field.So I don't see any of my tonics in there. But again, even nicely centered, these cells will go through my PS I continue, here's a good example. Here's a good example of what I would avoid that, see how bright that is?That's very bright. That's probably remnants of an apoptotic cell. And the reason I would avoid that is because since it's so bright, when the software auto focuses, it will autofocus just on that and it won't be in the same plane as all these other cells.And so I won't get any information from that field. That looks nice. There's a, this is a pro metaphase right here, That's A metaphase.So, and I'm gonna take three positions per well. So that was three positions and now I'm going to go to my next Well, so you can, so what I will probably do is take images every 10 minutes. So it's a compromise.Lifestyle imaging is always a compromise, right? You have to image, you have to keep the cells healthy, especially if you're interested in mitosis. So you have to minimize their exposure to the fluorescent line.And, but you wanna get information. So you have to take images, you have to, so you have to compromise, you have to make a choice about what you want to capture, what kind of information you wanna capture. For our purposes, mitosis tends to take about an hour.So if we're taking 10 minute intervals, that's pretty good. We see prophase, we see metaphase, we see anaphase, and we can, we can see when there are, there are treatments or drugs or siRNAs that affect those specific cell cycle stages. So for our purposes, that's enough, but we always want more.We always want more positions or finer time and interval or something like that. So it's a compromise. So, and then I'll hit start.And so our software, it does an autofocusing pass on the first pass. So once I hit start, it should start auto autofocusing and you'll see the shutter open, open and close between auto focusing. So I'll hit start.So that's the shutter opening and closing. The Z motor is engaging and it's actually trying to find the plane with the most contrast, which will be the most in focus. You can actually see on the screen going in and out of finding the best plane of focus that might be worrisome.So what it does is it, it drives the Z motor to find the best, the most in focus plane focus, well the most in image. And then it acquires picture there and then it learns that Z position. And then we'll use that Z position for the next 12 passes.And then we'll do the auto focusing pass again. What Kind of experiments do they do? Okay, so our lab is mostly interested in understanding what regulates mitic progression, or at least I'm most interested in understanding that.So with live cell imaging and especially our multi position, long-term imaging scope, you can take, you can film cells for two or three days entering anding mitosis, and you can image multiple rounds of mitosis so you can understand. And then you can put drugs or RNAs or any kind of perturbations on the cells that you would like to study. So you can understand how, how those drugs or, or genes are, are important in regulating mitotic progression.Because it's not fixed cell work and live cell work, you can understand what, when you need to have a perturbation to affect mitosis or what stage of mitosis is affected. So it's really powerful in that respect. What, what are the main technical elements of this experiments?What are the possible problems? Well, the biggest problem besides getting the rig set up, getting the rig set up is not trivial. A lot of people have the rig, but just making sure it works well for your application.The financial element is not, you know, is not small. So there's the setting up of the apparatus and then, and then once you gather the data, there's a real data analysis issue. It takes these movie, if, so, if you're gathering 40 movies each with 30 cells, so you have 40 in 40 fields that you just filmed and each of those fields has 30 cells, then you have to sit down with each one of those movies and look at each one of those cells in that movie.So it takes a really long time to do data analysis. And that's, so it takes a week to analyze data that it took you two days to collect. So there's a, that's a very big obstacle in terms of gathering lots of data.So it's not, while it's high throughput data collection, it's not high throughput analysis. So what would you make to make this experiment really high throughput? So our lab is in the process of developing a program that does the data analysis automatically.And it's a pretty decent program. It's not perfect. The human eye is always better, but it's getting there.So What are the interesting application of this experiment? Maybe other fields you can mention? Besides cell division?You mean for the, oh, for the live cell imaging? Yes.Well, you can do cell motility assays. You can, depending on the, the microscopy that's involved, you can, you know, look at how cells, you could look at tumor development if you really wanted to.But there, you, you're, if as long as you have the right imaging set up, you can, you're pretty much not limited in terms of anything. Our scope happens to be just a wide field and we use 20 by 20 x objective. So it's a pretty low resolution set up, but that's sufficient for what we need to see.So there are, but there definitely aren't, you wouldn't be limited. Anything that has any dynamic kind of experiment, which is biology, it would be, would really benefit from lifestyle imaging. So calcium imaging, although you couldn't do it on our apparatus, that's a live cell.You definitely need live cell imaging for that. Like I said, patient assays, motility assays, those kinds of things would really benefit from live cell imaging. Would you like to tell a few words about your previous experience?How long do you work on these techniques? What do you you work on before? Wow.So in my PhD I didn't work.I did a lot of confocal microscopy, but no lifestyle imaging. And so during my postdoc I was, I helped Dr.King get this apparatus that, that we just used to set up these fragment, get that up and running. And, and so during my postdoc, I had a lot of experience with both widefield with TimeLapse imaging on our scope, which is the widefield and also TimeLapse imaging with confocal microscopy.And I would say that it's really powerful. You get a lot of information, but it's not easy to get working perfectly. So it really requires a lot of attention and a lot of attention to details at every little step of the process.And, and then you still have problems. So it's a tough application, but you can really get, if, if it's what you need to have to, to study your problem, your scientific problem, then it, it's invaluable in that respect, but it's tough. So find someone who knows what they're doing and use them.That's my advice. So good.

长期使用宽视场显微镜的成像哺乳动物细胞

科研

生物学

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

多层安装于斑马鱼的长期光表镜

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

ATP合酶二聚体线粒体通过冷冻电子断层扫描可视化

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

体内转染和基因敲除在脊髓通过快速无创腰椎鞘内注射小鼠

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber2. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.

低温电子显微镜样品制备借助于聚焦离子束

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell&#8217;s ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue.

Here, we introduce a method, cocem3D, to unveil the ultrastructure of a specific cell in its native tissue by bridging confocal and serial block-face scanning electron microscopy.

一个具体的感觉细胞的共聚焦相关和3D电子显微镜

Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糖皮质激素粪分析:在马属动物无创监测肾上腺

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.