Name: delivery of exogenous artificially synthesized mirna mimic to the kidney using polyethylenimine nanoparticles in several kidney disease mouse models
Uploaded: 2022-05-10T14:00:00
Description: Watch this Scientific Journal Video about Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models at JoVE.c

这项工作得到了JSPS KAKENHI的部分支持（批准号21K08233）。我们感谢Edanz （https://jp.edanz.com/ac）编辑这份手稿的草稿。

所有动物实验方案均经吉智医科大学动物伦理委员会批准，并按照《吉智医科大学实验动物指南》中的实验动物使用和护理指南进行。在这里，我们证明了miRNA模拟递送到肾脏，导致其使用UUO小鼠过表达。本研究已获得吉智医科大学伦理委员会批准[肾纤维化批准号19-12，急性肾脏感染（AKI）批准17-024，糖尿病肾病批准19-11]。
1. PEI-NPs-miRNA模拟复合物的制备
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<ol>
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(163), e61378 (2020).</li><li>Kushibiki, T., Nagata-Nakajima, N., Sugai, M., Shimizu, A., Tabata, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+anti-fibrotic+activity+of+plasmid+DNA+expressing+small+interference+RNA+for+TGF-beta+type+II+receptor+for+a+mouse+model+of+obstructive+nephropathy+by+cationized+gelatin+prepared+from+different+amine+compounds.">Enhanced anti-fibrotic activity of plasmid DNA expressing small interference RNA for TGF-beta type II receptor for a mouse model of obstructive nephropathy by cationized gelatin prepared from different amine compounds.</a> Journal of Controlled Release: Official Journal of the Controlled Release Society. 110 (3), 610-617 (2006).</li><li>Xia, Z., 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=Suppression+of+renal+tubulointerstitial+fibrosis+by+small+interfering+RNA+targeting+heat+shock+protein+47.">Suppression of renal tubulointerstitial fibrosis by small interfering RNA targeting heat shock protein 47.</a> American Journal of Nephrology. 28 (1), 34-46 (2008).</li><li>Tanaka, T., 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=In+vivo+gene+transfer+of+hepatocyte+growth+factor+to+skeletal+muscle+prevents+changes+in+rat+kidneys+after+5/6+nephrectomy.">In vivo gene transfer of hepatocyte growth factor to skeletal muscle prevents changes in rat kidneys after 5/6 nephrectomy.</a> American Journal of Transplantation: Official Journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 2 (9), 828-836 (2002).</li><li>Hamar, P., 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=Small+interfering+RNA+targeting+Fas+protects+mice+against+renal+ischemia-reperfusion+injury.">Small interfering RNA targeting Fas protects mice against renal ischemia-reperfusion injury.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (41), 14883-14888 (2004).</li><li>Ma, D., 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=Xenon+preconditioning+protects+against+renal+ischemic-reperfusion+injury+via+HIF-1alpha+activation.">Xenon preconditioning protects against renal ischemic-reperfusion injury via HIF-1alpha activation.</a> Journal of the American Society of Nephrology: JASN. 20 (4), 713-720 (2009).</li><li>Wei, 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=Short+hairpin+RNA+knockdown+of+connective+tissue+growth+factor+by+ultrasound-targeted+microbubble+destruction+improves+renal+fibrosis.">Short hairpin RNA knockdown of connective tissue growth factor by ultrasound-targeted microbubble destruction improves renal fibrosis.</a> Ultrasound in Medicine and Biology. 42 (12), 2926-2937 (2016).</li></ol>

使用本手稿中提出的方案，PEI-NPs可以将miRNA模拟物递送到肾脏以诱导靶miRNA的过表达，从而在几种肾脏疾病的 体内小鼠模型中 产生治疗效果，包括肾纤维化，糖尿病肾病和AKI。
制备PEI-NPs和miRNA模拟物复合物的方法非常简单。PEI-NPs的带正电荷的表面在刚刚混合13，14，15，16，<sup class=...

miRNA，即未翻译成蛋白质的小非编码RNA（21-25个碱基），通过破坏它们在各种肾脏疾病中的翻译来抑制大量靶信使RNA（mRNA）1，2。因此，采用外源性人工合成的miRNA模拟物或抑制剂的基因治疗是抑制许多肾脏疾病发展的潜在新选择3，4，5。
尽管miRNA模拟物或抑制剂有望用于基因治疗，但递送到靶器官仍然是体内实验开发其临床潜力的一大障碍。由于人工合成的miRNA模拟物或抑制剂会立即被血清RNA酶降解，因此在体内全身施用时，它们的半衰期缩短6。此外，如果没有合适的载体，miRNA模拟物或抑制剂穿过质膜和转染细胞质的效率通常要低得多7，8。这些证据表明，需要开发miRNA模拟物或肾脏抑制剂递送系统，使其能够在临床环境中使用，并使其成为各种肾脏疾病患者的新治疗选择。
病毒载体已被用作载体，将外源性miRNA模拟物或抑制剂递送到肾脏9，10。尽管病毒载体是为生物安全性和转染功效而开发的，但仍可能导致干扰素反应和/或遗传不稳定11，12。为了克服这些问题，我们在几种肾脏疾病小鼠模型中使用聚乙烯亚胺纳米颗粒（PEI-NPs）开发了miRNA模拟肾脏递送系统13，14，15。
PEI-NPs是基于线性聚合物的NPs，可以有效地将寡核苷酸（包括miRNA模拟物）递送到肾脏，并且由于其长期安全性和生物相容性，被认为是制备非病毒载体的首选13，16，17。
本研究证明了系统外源miRNA通过尾静脉注射模拟PEI-NPs递送对单侧输尿管梗阻（UUO）产生的肾纤维化模型小鼠的影响。此外，我们证明了系统外源miRNA模拟物通过尾静脉注射与PEI-NPs递送在糖尿病肾病模型小鼠（db / db小鼠：C57BLKS / J Iar -+Lepr db / + Leprdb）和由肾缺血再灌注损伤（IRI）产生的急性肾损伤模型小鼠中的影响。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4&#8217;,6-diamidino-2-phenylindole for staining to nucleus</td>
			<td>Thermo Fisher Scientific</td>
			<td>D-1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer RPE</td>
			<td>Qiagen</td>
			<td>79216</td>
			<td>Wash buffer 2</td>
		</tr>
		<tr>
			<td>Buffer RWT</td>
			<td>Qiagen</td>
			<td>1067933</td>
			<td>Wash buffer 1</td>
		</tr>
		<tr>
			<td>Control-miRNA-mimic (artificially synthesized miRNA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>Not assigned</td>
			<td>5&#8217;-UUCUCCGAACGUGUCACGUTT- 3&#8217; (sense) 
			5&#8217;-ACGUGACACGUUCGGAGAATT-3&#8242; (antisense)</td>
		</tr>
		<tr>
			<td>Cy3-labeled double-strand oligonucleotides</td>
			<td>Takara Bio Inc.</td>
			<td>MIR7900</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein-labeled Lotus tetragonolobus lectin</td>
			<td>Vector Laboratories Inc</td>
			<td>FL-1321</td>
			<td></td>
		</tr>
		<tr>
			<td>In vivo-jetPEI</td>
			<td>Polyplus</td>
			<td>101000021</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp Optical 96-well reaction plate for qRT-PCR</td>
			<td>Thermo Fisher Scientific</td>
			<td>4316813</td>
			<td>96-well reaction plate</td>
		</tr>
		<tr>
			<td>MicroAmp Optical Adhesive Film</td>
			<td>Thermo Fisher Scientific</td>
			<td>4311971</td>
			<td>Adhesive film for 96-well reaction plate</td>
		</tr>
		<tr>
			<td>miRNA-146a-5p mimic (artificially synthesized miRNA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>Not assigned</td>
			<td>5&#8217;-UGAGAACUGAAUUCCAUGGGU 
			UT-3&#8242; (sense) 5&#8217;-CCCAUGGAAUUCAGUUCUCAUU -3&#8242; (antisense)</td>
		</tr>
		<tr>
			<td>miRNA-146a-5p primer</td>
			<td>Qiagen</td>
			<td>MS00001638</td>
			<td>Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021)</td>
		</tr>
		<tr>
			<td>miRNA-181b-5p mimic (artificially synthesized miRNA)</td>
			<td>Gene design</td>
			<td>Not assigned</td>
			<td>5&#8217;-AACAUUCAUUGCUGUCGGUGG 
			GUU-3&#8217;</td>
		</tr>
		<tr>
			<td>miRNA-181b-5p primer</td>
			<td>Qiagen</td>
			<td>MS00006083</td>
			<td>Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021)</td>
		</tr>
		<tr>
			<td>miRNA-5100-mimic (artificially synthesized miRNA)</td>
			<td>Gene design</td>
			<td>Not assigned</td>
			<td>5&#8217;-UCGAAUCCCAGCGGUGCCUCU -3&#8242;</td>
		</tr>
		<tr>
			<td>miRNA-5100-primer</td>
			<td>Qiagen</td>
			<td>MS00042952</td>
			<td>Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021)</td>
		</tr>
		<tr>
			<td>miRNeasy Mini kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td>Membrane anchored spin column in a 2.0-mL collection tube</td>
		</tr>
		<tr>
			<td>miScript II RT kit</td>
			<td>Qiagen</td>
			<td>218161</td>
			<td>Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021)</td>
		</tr>
		<tr>
			<td>miScript SYBR Green PCR kit</td>
			<td>Qiagen</td>
			<td>218073</td>
			<td>Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021)</td>
		</tr>
		<tr>
			<td>QIA shredder</td>
			<td>Qiagen</td>
			<td>79654</td>
			<td>Biopolymer spin columns in a 2.0-mL collection tube</td>
		</tr>
		<tr>
			<td>QIAzol Lysis Reagent</td>
			<td>Qiagen</td>
			<td>79306</td>
			<td>Phenol/guanidine-based lysis reagent</td>
		</tr>
		<tr>
			<td>QuantStudio 12K Flex Flex Real-Time PCR system</td>
			<td>Thermo Fisher Scientific</td>
			<td>4472380</td>
			<td>Real-time PCR instrument</td>
		</tr>
		<tr>
			<td>QuantStudio 12K Flex Software version 1.2.1.</td>
			<td>Thermo Fisher Scientific</td>
			<td>4472380</td>
			<td>Real-time PCR instrument software</td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td>Qiagen</td>
			<td>129112</td>
			<td></td>
		</tr>
		<tr>
			<td>RNU6-2 primer</td>
			<td>Qiagen</td>
			<td>MS00033740</td>
			<td>Not available because Qiagen has changed qRT-PCR kits (from miScript miRNA PCR system to miRCURY LNA miRNA PCR System from May 2021)</td>
		</tr>
		<tr>
			<td>Tissue-Tek OCT (Optimal Cutting Temperature Compound)</td>
			<td>Sakura Finetek Japan Co.,Ltd.</td>
			<td>Not assigned</td>
			<td></td>
		</tr>
	</tbody>
</table>

delivery of exogenous artificially synthesized mirna mimic to the kidney using polyethylenimine nanoparticles in several kidney disease mouse models

microRNA（miRNA）是未翻译成蛋白质的小非编码RNA（21-25个碱基），通过破坏和抑制它们在各种肾脏疾病中的翻译来抑制大量靶信使RNA（mRNA）。因此，通过外源人工合成的miRNA模拟物交替表达miRNA是抑制许多肾脏疾病发展的潜在有用治疗选择。然而，由于血清RNA酶会立即降解体内系统施用的外源性miRNA模拟 物，因此将miRNA递送到肾脏仍然是一个挑战。因此，能够保护外源性miRNA模拟物免受RNA酶降解并显着将其递送到肾脏的载体是必要的。许多研究使用病毒载体将外源性miRNA模拟物或抑制剂递送到肾脏。然而，病毒载体可能导致干扰素反应和/或遗传不稳定。因此，病毒载体的开发也是临床使用外源性miRNA模拟物或抑制剂的障碍。为了克服这些关于病毒载体的担忧，我们开发了一种非病毒载体方法，使用尾静脉注射聚乙烯亚胺纳米颗粒（PEI-NPs）将miRNA模拟物递送到肾脏，这导致靶miRNA在几种肾脏疾病小鼠模型中显着过表达。

根据微阵列、qRT-PCR 和/或基因治疗应用的数据库研究，选择了下面描述的用于肾纤维化、糖尿病肾病和 AKI 的靶 miRNA。有关更多详细信息，请参阅以前的出版物13，14，15。
使用PEI-NPs的miRNA-146a-5p模拟物在肾纤维化小鼠中的递送和作用13 荧光显微镜分析表明，尾?...

Watch this Scientific Journal Video about Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models at JoVE.c

Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models

在这里，我们通过在几种肾脏疾病小鼠模型中通过尾静脉注射非病毒载体和聚乙烯亚胺纳米颗粒 将 外源人工合成的miRNA模拟物输送到肾脏。这导致靶miRNA在肾脏中的显着过表达，导致几种小鼠模型中肾脏疾病的进展受到抑制。

在几种肾脏疾病小鼠模型中使用聚乙烯亚胺纳米颗粒将外源人工合成的miRNA模拟物递送到肾脏

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by ...

这种方法可以帮助回答治疗肾脏microRNA的关键问题，这仍然是一个挑战。这种技术的主要优点是，它能够模拟肾脏的microRNA治疗，而不会引起干扰素反应或遗传不稳定。首先将10微升PEI-NPs溶解在1.5毫升微量离心管中的90微升5%葡萄糖溶液中。然后轻轻涡旋管子并向下旋转。将溶解在100微摩尔无核酸酶水中的50微升人工合成的miRNA与1.5毫升微量离心管中的50微摩尔10%葡萄糖溶液混合。轻轻涡旋管并向下旋转。该过程产生溶解在5%葡萄糖溶液中的miRNA。然后将100微升PEI-NPs与5%葡萄糖溶液混合，将100微升miRNA模拟物与5%葡萄糖溶液混合。轻轻涡旋管并向下旋转。将混合物在室温下孵育15分钟，以制备稳定的PEI-NPs miRNA模拟物复合物。将两微升PEI-NPs溶解在1.5毫升微量离心管中的98微升10%葡萄糖溶液中。轻轻涡旋管并向下旋转。然后将100微升Cy3标记的miRNA模拟物加入10%葡萄糖溶液中的100微升PEI-NPs中，并轻轻涡旋试管。将混合物在室温下孵育15分钟，以制备稳定的PEI-NPs-Cy3标记的miRNA模拟物复合物。将UUO小鼠头先放入50毫升离心管中，无需麻醉，然后将尾巴穿过盖子上准备好的孔。用30号针头填充1毫升注射器，用200微升PEI-NPs-Cy3标记的miRNA模拟复合物填充复合物，并通过小鼠尾静脉注射复合物。接下来，用手术剪刀和镊子切开皮肤、肌肉和肋骨，露出心脏。在右心房切开后，将PBS注射到左心室，直到肾脏颜色变为淡黄色。这表明整个鼠标主体都注入了 PBS。收集肾脏后，取出肾囊，并用PBS清洗两次。将肾组织样品嵌入OCT化合物中，并在液氮中冷冻。使用低温恒温器制备切片并将切片安装到硅烷涂层载玻片上。用4%多聚甲醛固定它们，并用PBS轻轻清洗载玻片两次。最后，通过荧光显微镜在100x和400x放大倍率下以及适当的成像软件可视化荧光染色。代表性图像显示了PEI-NPs-miRNA模拟复合物的结构。这些图像描述了使用PEI-NPs的miRNA-146a-5p-模拟物在肾纤维化中的递送和作用。荧光显微镜分析表明，尾静脉注射PEI-NPs可将miRNA模拟物递送至肾小管间质间隙。在UUO产生的肾纤维化小鼠中，这包括肾小管细胞，其在肾脏和肾小球中没有规则形式。qRT-PCR分析显示，注射PEI-NPs-miRNA-146a-5p-模拟物诱导miRNA-146a-5p在肾脏中显著过表达。此处显示了天狼星红色染色的组织学分析。荧光显微镜分析表明，尾静脉注射PEI-NPs可将miRNA模拟物递送至糖尿病肾模型小鼠体内肾脏的肾小球和肾小管间质间隙。此外，qRT-PCR分析显示，注射PEI-NPs-miRNA-181b-5p-模拟物诱导肾脏中miRNA-181b-5p的显着过表达。高碘酸-希夫染色的组织学分析表明，从10至20周龄每周注射一次PEI-NPs-miRNA-181b-5p-模拟物可抑制小鼠糖尿病肾病的进展。此处显示了PEI-NPs-miRNA-5100-模拟物在缺血再灌注损伤产生的急性肾损伤模型小鼠中的递送和作用。这些图像描述了荧光显微镜分析qRT-PCR分析的数据，以评估注射PEI-NPs-miRNA-5100-模拟物后miRNA-5100的过表达效应，以及苏木精-伊红染色肾脏的组织学分析。在缺血再灌注损伤（IRI）手术前两天通过尾静脉注射PEI-NPs可防止IRI诱导的急性肾损伤进展。执行此技术时，准备五微米厚的切片，其中包括尽可能多的肾组织。您还可以准备七微米厚的切片。

Research

JoVE Journal

Medicine

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

小鼠胰岛的分离和包膜下肾移植的方法

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

科研

医学

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

光谱核型多囊肾病的研究在人类和小鼠的染色体异常

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Assessment of Vascular Function in Patients With Chronic Kidney Disease

血管功能在慢性肾脏疾病的评估

Patients with chronic kidney disease (CKD) have significantly increased risk of cardiovascular disease (CVD) compared to the general population, and this ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

多囊肾一种可能的斑马鱼型号：击倒<em&gt; Wnt5a的</em&gt;原因在斑马鱼肾囊肿

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Mouse Kidney Transplantation: Models of Allograft Rejection

鼠肾移植：移植排斥反应模型

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

在多囊肾小型啮齿动物模型采用超高场磁共振成像<em&gt;在体内</em&gt;表型和药物监测

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Murine Kidney Transplant Technique

小鼠肾脏移植技术

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

常温<em&gt;离体</em&gt;肾脏的灌注肾的移植保护移植前

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

Intravascular Delivery of Biologics to the Rat Kidney

生物血管内送货到大鼠肾脏

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

Assessment of Kidney Function in Mouse Models of Glomerular Disease

小鼠肾小球疾病模型肾脏功能的评价

The use of murine models to mimic human kidney disease is becoming increasingly common. Our research is focused on the assessment of glomerular function ...