Name: purification and transplantation of myogenic progenitor cell derived exosomes to improve cardiac function in duchenne muscular dystrophic mice
Uploaded: 2019-04-10T14:00:00
Description: Watch this Scientific Journal Video about Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice at JoVE.com

唐得到了美国心脏协会的部分支持: GRNT31430008、NIH-AR070029、NIH-HL086555、NIH-HL134354。

动物是根据奥古斯塔大学佐治亚医学院动物护理和使用机构委员会批准的协议和动物福利条例处理的。
1. mpc 衍生的外显子的分离和纯化
<ol><li>种子 5 x10 6 c2c12 细胞在一个15厘米的细胞培养盘与20毫升完成 dulbecco 的改性鹰的培养基 (dmem) 含有10% 的胎儿牛血清 (fbs), 100 Uml 青霉素 g 和100μgml 链霉素。在37°c 和 5% co 2 下孵化.</li>
	<li>利用摆动的桶式转子, 在 100, 000 <...

<ol>
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分离纯外质体的方法对于研究外显子的功能是必不可少的。外显子体分离的常用技术之一是聚乙二醇介导的沉淀17,18,25。外显子可以沉淀在 Peg 中, 并通过低速离心颗粒。Peg 介导的纯化非常方便, 成本低, 它不需要任何先进的设备, 但人们担心外显子的纯度, 因为其他脂蛋白可能沉淀在一起, 很难去除。超滤是一种常规的外显子分?...

杜氏肌营养不良症 (DMD) 是一种 x 连锁隐性、进行性神经肌肉疾病, 由 DMD 基因突变和功能性营养不良蛋白 1的丧失引起。肌营养蛋白主要表现在骨骼肌和心肌中, 在平滑肌、内分泌腺体和神经元2,3中表达较少。Dmd 是最常见的肌肉营养不良症, 发病率为每3, 500 至 5,000名新生男孩1人。个人通常会出现进行性肌肉坏死, 在青春期早期失去独立行走, 以及在生命的第二至第三年死于心力衰竭和呼吸衰竭.
扩张型心肌病、心律失常和充血性心力衰竭是 dmd7,8的常见心血管表现。这种疾病无法治愈, 支持性治疗可能会改善症状或延缓心力衰竭的进展, 但很难改善心脏功能9,10.
与 DMD 患者类似, x 连锁肌营养不良症 (MDX) 小鼠缺乏营养不良蛋白, 并出现心肌病11的症状, 因此被广泛用于 DMD 相关心肌病的研究。为了恢复受影响肌肉中的营养不良蛋白, 异基因干细胞治疗已被证明是治疗 dmd12、13、14的有效方法。外体, 30-150nm 膜囊泡分泌的各种细胞类型, 发挥关键作用, 通过遗传物质运输细胞间的通信, 如信使 rna (mrna) 和非编码 rna15,16,17 ,18,19,20,21。
我们以前的研究表明, 从肌源性祖细胞 (MPC) 中提取的外显子, 如 C2C12 细胞系, 可以转移营养不良素 mRNA 到宿主心肌细胞后, 直接心脏注射22, 这表明同种异体传递Mpc 衍生外显子 (MPC-Exo) 可以短暂恢复 MDX 小鼠的 DMD 基因表达。本文的重点是 MPC-Exo 纯化和移植技术。

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			<td height="21" style="height:21px;width:424px;">0.22-&#956;m Filter</td>
			<td style="width:280px;">Fisherbrand</td>
			<td style="width:185px;">09-720-004</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-cm Cell Culture Dish</td>
			<td>Thermo Fisher Scientific</td>
			<td>157150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-gauge catheter</td>
			<td style="width:280px;">TERUMO</td>
			<td>SR-OX2419CA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">31-gauge insulin needle</td>
			<td style="width:280px;">BD</td>
			<td>328291</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4% paraformaldehyde&#160;</td>
			<td style="width:280px;">Affymetrix</td>
			<td>AAJ19943K2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL Centrifuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-0 suture</td>
			<td style="width:280px;">Pro Advantage by NDC</td>
			<td>P420697</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 goat anti-rabbit IgG</td>
			<td style="width:280px;">Thermo Fisher Scientific</td>
			<td>A-11008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antibiotic Antimycotic Solution</td>
			<td>Corning&#160;</td>
			<td>30-004-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Dystrophin antibody</td>
			<td style="width:280px;">Abcam</td>
			<td>ab15277</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antigen retriever&#160;</td>
			<td style="width:280px;">Aptum Biologics</td>
			<td>R2100-US</td>
			<td>Antigen recovery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Autofluorescence Quenching Kit&#160;</td>
			<td style="width:280px;">Vector Laboratories</td>
			<td>SP-8400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C2C12 cell line</td>
			<td>ATCC</td>
			<td>CRL-1772</td>
			<td></td>
		</tr>
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			<td height="25" style="height:25px;">Centrifuge</td>
			<td>Unico</td>
			<td>C8606</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Change-A-Tip High Temp Cauteries</td>
			<td>Bovie Medical Corporation</td>
			<td>HIT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscopy</td>
			<td style="width:280px;">Zeiss</td>
			<td>Zeiss 780 Upright Confocal</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">DBA/2J-mdx mice</td>
			<td>The Jackson Laboratory</td>
			<td>013141</td>
			<td></td>
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			<td height="21" style="height:21px;">DMEM</td>
			<td>Corning&#160;</td>
			<td>10-013-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum (FBS)</td>
			<td>Corning&#160;</td>
			<td>35-011-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat serum&#160;</td>
			<td style="width:280px;">MP Biomedicals, LLC</td>
			<td>191356</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane</td>
			<td style="width:280px;">Patterson Veterinary</td>
			<td>07-893-1389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine</td>
			<td style="width:280px;">Henry Schein</td>
			<td>056344</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mounting Medium with DAPI&#160;</td>
			<td style="width:280px;">Vector Laboratories</td>
			<td>H-1500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Retractor Set</td>
			<td>Kent Scientific</td>
			<td>SURGI-5001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene glycol tert-octylphenyl ether</td>
			<td style="width:280px;">Fisher Scientific</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rodent ventilator</td>
			<td style="width:280px;">Harvard Apparatus</td>
			<td>55-7066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW-28 Ti rotor</td>
			<td>Beckman</td>
			<td>342207</td>
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			<td height="21" style="height:21px;">The Vevo 2100 Imaging Platform</td>
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			<td>Vevo 2100</td>
			<td>Ultrasound System&#160;</td>
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			<td height="21" style="height:21px;">Ultracentrifuge</td>
			<td>Beckman</td>
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			<td height="21" style="height:21px;">Ultra-Clear Tubes</td>
			<td>Beckman</td>
			<td>344058</td>
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			<td height="21" style="height:21px;">Xylazine (XylaMed)</td>
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</table>

purification and transplantation of myogenic progenitor cell derived exosomes to improve cardiac function in duchenne muscular dystrophic mice

杜琴肌肉营养不良 (DMD) 是一种由缺乏功能性营养不良蛋白引起的 x 连锁隐性遗传病。这种疾病无法治愈, 随着病情的发展, 患者会出现扩张型心肌病、心律失常和充血性心力衰竭的症状。DMD Mdx突变体小鼠不表达营养不良蛋白, 常被用作 DMD 的小鼠模型。在我们最近的研究中, 我们观察到大类型 (WT)-肌原体细胞衍生外显子 (MPC-Exo) 的心肌内注射短暂地恢复了 DMDMdx突变体小鼠心肌中营养不良蛋白的表达, 这与之相关心脏功能的短暂改善表明 WT-MPC-Exo 可以提供一个选项来缓解 DMD 的心脏症状。本文介绍了 MPC-Exo 纯化并移植到dmd Mdx 突变体小鼠心脏中的技术。

图 1a 显示了从 c2c12 细胞中分离和纯化外显体的流程图。为了确认外显子的存在, 我们进行了透射电子显微镜分析。透射电子显微镜图像 (图 1b) 显示了 c2c12 衍生外显子的明亮和圆形囊泡的形态。西方印迹分析证实存在外显子标记, 包括 CD63 和 TSG101 (图 1c)。
我们观察到心?...

Watch this Scientific Journal Video about Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice at JoVE.com

Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice

在这里, 我们提出了一个方案, 以过渡改善心脏功能的杜氏肌营养不良小鼠移植从正常的肌原细胞外周肌。

肌源性祖细胞衍生外质细胞的纯化与移植, 改善杜氏肌营养不良性小鼠的心功能

Duchene Muscular Dystrophy (DMD) is an X-linked recessive genetic disease caused by a lack of functional dystrophin protein. The disease cannot be cured, ...

该协议提出了通过移植来自正常肌原细胞的外生细胞，过渡性地改善Duchenne肌肉营养不良小鼠心脏功能的方法。外体移植可以改善DMD小鼠的心脏功能，同时增加接受者心脏的营养不良表现。适当的外向分离、纯化和心肌内移植方法有助于外向移植的成功，以改善患有Duchenne肌肉萎缩症的小鼠的心脏功能。MPC衍生的外体疗法可能通过外显体中显性正常肌营养不良mRNA转移来改善骨骼肌功能。本视频演示了外显体纯化、心肌内外显体传递和移植后超声心动图的新技术。超声心动图的演示将由我们实验室的技术人员严申完成。为了开始这个程序，将500万C2C12细胞播种到15厘米培养皿中，含有20毫升完整的DMEM，含有10%FBS和抗生素。在37摄氏度下孵育，二氧化碳含量为5%。接下来，使用摆动桶转子在10万次G和4摄氏度的超离心FBS18小时，以准备外体耗尽的介质。丢弃颗粒并收集上一液。当单层细胞在培养皿中达到80%的汇合时，用外显性培养基替换完整的DMEM。使用转移移液器每 48 小时从细胞培养盘收集上流水。将管子在150倍G下离心10分钟，以取出剩余的细胞。通过 0.22 微米过滤器过滤上清液，消除任何细胞碎屑，并过滤中到超透明管。接下来，使用回转桶转子在 100，00 倍 G 和 4 摄氏度的管上超离心 120 分钟，以沉淀外体。在PBS中重新填充含有外显颗粒的颗粒，然后用PBS填充整个超透明管。超离心这种悬浮液在100，00倍G和4摄氏度120分钟，以消除污染蛋白质。丢弃上一杯，在100微升PBS中重新暂停外显颗粒。储存在零下80摄氏度，供将来使用。首先，用胶带固定每个肢体，将每只麻醉小鼠固定到手术平台上的上肢位置。水平在上牙下方放置 3-0 缝合线，将上颚保持到位。接下来，在小鼠胸部左侧涂抹脱毛霜，以去除皮肤上的冷杉。使用 24 量表导管，通过口腔进行气管插管。使用啮齿动物呼吸机以每分钟 195 次呼吸的速度用室内空气为鼠标通风。用70%酒精和10%的碘消毒皮肤。然后从左尾边缘到左窝，进行 10 到 15 毫米的斜切口。用剪刀剪小胸腔和小胸腔。并通过第四个海岸间空间进行左胸腔。轻轻插入缩回带，将胸腔扩散到 10 毫米的宽度，注意不要损坏左肺。在此之后，使用两个直钳子去除佩里卡。将它们拉开，并把它们放在缩回器尖端后面，以暴露心脏。使用 31 量胰岛素针将 MPC-Exo 或 PBS 在一个站点的左心室前壁中注射 MPC-Exo 或 PBS。然后，使用6-0尼龙缝合线，以关闭胸腔，胸腔肌肉和皮肤，顺序。PBS外体移植两天后，对小鼠进行麻醉，如文本协议中所述。使用胶带将鼠标固定在超声位置，并在左胸区域涂抹预热声学凝胶。接下来，使用超声心动图评估左心室功能。以两个维度获取左心室的准体长轴视图，然后旋转超声波探头 90 度，以获得左心室短轴视图在子宫颈肌肉水平。之后，记录M模式超声心动图图像并测量左心室端膜直径、左心室端膜收缩体积、左心室端膜体积和左心室端膜收缩体积。对于心脏测量功能，小鼠的心率应控制至少400 bpm。从C2C12细胞分离和纯化外体后，通过透射电子显微镜分析确认其存在。透射电子显微镜图像显示了C2C12衍生外体明亮圆形囊泡的形态。西方印迹分析证实存在外显性标记，包括CD63和茨格101。在心肌注射到mdx小鼠左心室前壁后观察到半透明水肿区，这表明注射到心肌中是成功的。然后进行肌营养不良素的免疫荧光染色，以确定心脏MPC-Exo分娩是否恢复mdx心脏中的肌营养不良蛋白表达。部分恢复肌营养不良素表达观察与膜定位在一些心肌细胞。心脏功能在心肌分娩两天后通过超声心动图进行测量，以确定MPC-Exo的转增是否改善了mdx小鼠的心脏功能。与PBS相比，MPC-Exo治疗可以改善内壁运动，这表明MPC-Exo移植改善了mdx小鼠的心脏功能。使用31量表胰岛素针，尖端在20度左右，对于成功将大多数外显体传递到心肌中，并最大限度地利用注射外体照射心肌细胞至关重要。按照该程序，在MPC衍生外体注射后，可以通过免疫荧光染色检测肌营养不良素表达，表明心脏MPC衍生外体分娩恢复肌营养不良蛋白表达。这项技术将为心肌病治疗铺平道路，使用外体携带治疗遗传物质，包括DNA、mRNA、lncRNA或微RNA。

Research

JoVE Journal

Genetics

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

科研

遗传学

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

CRISPR/Cas9-based 跳跃的原始生殖细胞移植在爪

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

支气管肺泡灌洗外来体在脂多糖致脓毒性肺损伤中的作用

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high ...

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

一种组合单细胞法表征人茎和祖种群的分子和免疫表型异质性

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines

基于多重 CRISPR 增强器的细胞系干扰增强功能解剖

Multiple enhancers often regulate a given gene, yet for most genes, it remains unclear which enhancers are necessary for gene expression, and how these ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

人牙小泡干细胞的分离、鉴定及微 rna 遗传修饰

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

非人类灵长类动物造血干细胞和祖细胞的制备及基因修饰

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...