Name: preparation of plasma membrane vesicles from bone marrow mesenchymal stem cells for potential cytoplasm replacement therapy
Uploaded: 2017-05-18T15:00:00
Description: Watch this Scientific Journal Video about Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy at JoVE.com

这项研究得到了李嘉诚基金会，广东省高校“海洋工业绿色科技”，“中国自然科学基金”项目（http://www.nsfc.gov.cn/资助号：30971665， 81172894,81370925）和广东省教育厅（http://www.gdhed.edu.cn/ Grant No.cxzd1123）。

8至12周龄的BALB / c小鼠购自上海实验动物中心（中国上海），并在特定的无病原菌和空调动物设施中饲养。动物护理和实验程序符合汕头大学实验动物使用和护理指南。 装置装配<ol><li>为了确保无菌，使用前打开组织培养罩的紫外线30分钟。 </li><li>拧下一次性25毫米过滤器单元，并将设备的盖子和底部浸入装有75％乙醇的200毫升玻璃杯中30分钟。该单元由医用级聚丙烯制成。 ...

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Cytoplasm replacement therapy as proposed in this manuscript has unique advantages over other reported approaches such as gene, molecular, and cell therapy. PMVs generated from BMSCs encapsulate not only the products of stemness genes but also intact cellular organelles, which are essential to remedy the ageing phenotypes associated with senescence. When young cytoplasm is delivered to senescent cells, the malfunctioning mechanisms may gain a brief relief; at the same time, the epigenome could be reprogrammed and invigor...

<html>已经投入了大量的努力来建立基因，酶和细胞替代疗法的方法。这导致了巨大的突破，甚至临床应用1，2，3。最近，基于核转移技术的有争议的线粒体替代疗法被应用于老年妇女的体外受精或携带致死线粒体DNA突变4 。在年龄相关疾病中发现的缺陷，包括动脉粥样硬化，2型糖尿病，阿尔茨海默病和癌症，通常是多方面的。已经证明脂滴的积累;沉淀淀粉样蛋白;展开蛋白质在内质网中的保留;缺陷型蛋白酶体，自噬体和线粒体有助于这些疾病的发展或恶化“外套参照”&gt; 5,6,7,8,9,10,11。目前没有可用于直接修复细胞质和细胞器故障的机制，这导致衰老和衰老表型。 我们以前通过机械挤压哺乳动物细胞报道了生物质膜囊泡（PMVs） 12 。除了细胞核外，膜或细胞溶质中的组分（包括蛋白质和RNA）以及细胞器如线粒体都发现在PMV中。基本上，PMV可以被认为是一个微型的去核细胞。更重要的是，PMV与线粒体缺乏的Rho0细胞的融合在正常培养条件下恢复有丝分裂活性。这是关于成立的第一份报告为细胞质替代疗法建立潜在的有效途径。 骨髓间充质干细胞（BMSCs）是通常从骨髓产生并且在培养物中容易扩增的多能祖细胞。胚胎干细胞标记Oct4，Nanog和SOX2在MSCs 13中检测到低水平。端粒酶活性也是可测量的。此外，不存在共刺激分子和人白细胞抗原（HLA）II类分子以及MSC上的低HLA I类表达使其成为同种异体或“现成”使用的理想细胞再生医学和免疫调节应用14 。 在这里，我们描述如何通过挤出通过具有3-μm孔隙的聚碳酸酯膜来制备来自小鼠BMSC的PMV，确定线粒体的存在并使用confoc检查PMV中膜电位的维持通过离心法制备浓缩但不聚集的PMV，并进行PMVs 体内注射到小鼠的腓肠肌中。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>IsoporeTM membranes</td>
			<td>Millipore</td>
			<td>TSTP04700</td>
			<td>3 mm pore</td>
		</tr>
		<tr>
			<td>Disposable filter unit</td>
			<td>Xinya, Shanghai, China</td>
			<td>25 mm</td>
			<td>Medical grade polypropylene</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>BD</td>
			<td>328446</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>pN1-EGFP</td>
			<td>Clontech</td>
			<td>&#160;6085-1</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker</td>
			<td>Molecular Probes</td>
			<td>M7514</td>
			<td>Green FM, 1 &#956;M</td>
		</tr>
		<tr>
			<td>JC-1</td>
			<td>Beyotime, Haimen, China</td>
			<td>C2006</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>CM-DiI</td>
			<td>Beyotime, Haimen, China</td>
			<td>C1036</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>Sigma</td>
			<td>P3143</td>
			<td>Mn = 75000</td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Nikon</td>
			<td>Eclipse TE 2000</td>
			<td>With CCD camera</td>
		</tr>
		<tr>
			<td>Confocol Microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM 510 Meta</td>
			<td></td>
		</tr>
		<tr>
			<td>PolyJet</td>
			<td>SigaGen</td>
			<td>SL100688</td>
			<td>For cell transfection</td>
		</tr>
	</tbody>
</table>

preparation of plasma membrane vesicles from bone marrow mesenchymal stem cells for potential cytoplasm replacement therapy

我们以前报道了通过机械挤压哺乳动物细胞产生质膜囊泡（PMV）。 PMV与线粒体缺陷Rho0细胞的融合在正常培养条件下恢复有丝分裂活性。据报道，动脉粥样硬化，2型糖尿病，阿尔茨海默病和癌症是与各种细胞类型的细胞溶质和细胞器中的多种机械和功能缺陷相关的年龄相关疾病。骨髓间充质干细胞（BMSCs）代表骨髓中独特的细胞群，具有自我更新能力，同时保持其多能性。通过PMV融合补充来自自体骨髓基质干细胞的年轻细胞质的衰老细胞提供了改善或甚至逆转年龄相关表型的有希望的方法。该方案描述了如何通过使用3的聚碳酸酯膜挤出从BMSC制备PMV1; m孔，确定线粒体的存在，并使用共聚焦显微镜检查PMV内膜电位的维持，通过离心浓缩PMV，并进行体内注射PMV至小鼠腓肠肌。

成功准备PMV的关键在很大程度上取决于过滤器单元的正确组装（ 图1 ），可以通过将1mL PBS推过膜来测试。如果发生泄漏，重新组装过滤器单元并再次测试。然而，当细胞被推过膜时，泄漏只能被可靠地测试。如果在常规显微镜下仅使用10X物镜检测到几个PMV，或者如果PMV的大小大多为1μm左右，则表明大多数单元由于组装不正确而被捕获在单元内部。尺寸在约3μ...

Watch this Scientific Journal Video about Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy at JoVE.com

Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

与年龄相关的疾病与细胞质组分中的多种缺陷有关。在这里，我们提出了从骨髓间充质干细胞制备质膜囊泡的方案。这种技术可能被用作细胞质替代疗法的手段，以改善或甚至逆转年龄相关表型。

从骨髓间充质干细胞制备血浆膜囊泡用于潜在的细胞质替代治疗

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs ...

Research

JoVE Journal

Developmental Biology

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

人类骨髓间充质干细胞的分离及其培养的多​​孔骨基质

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

科研

发育生物学

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

酶法从凝块骨髓样本拯救间充质干细胞

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

从人胎盘组织源分离和间充质干扩张/基质细胞

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

从人骨髓分离Mesangiogenic祖细胞（多用途储值卡）

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

从详细的空间表达图谱的动态表达数据的时间排序

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

从人类脐带和胎盘胎儿分离和鉴定间充质干细胞

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

水平整体安装：厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Single-cell Photoconversion in Living Intact Zebrafish

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Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...