Name: isolation and characterization of extracellular vesicles from adult schistosoma japonicum
Uploaded: 2018-05-22T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum at JoVE.com

本研究部分或全部由中国国家自然科学基金 (31472187 和 31672550) 和中国科学院农业科技创新项目资助。

所有动物实验均获中国科学院上海兽医研究所地方伦理委员会批准 (许可证编号: SHVRIAU-14-0101)。
1.血吸虫的体外区域性
注意:血吸虫表示生物危害。在处理蠕虫、血吸虫悬浮液或其他相关生物材料时, 工人应始终佩戴乳胶手套。新西兰兔感染了2000尾蚴通过腹部暴露。血吸虫在肝脏阶段从感染了日本血吸虫尾蚴的?...

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最近对电动汽车的研究表明, 血吸虫电动车在宿主病原体相互作用中起着重要作用3,9,12,16。为了进一步解决它们的管理功能, 必须将 EVs 与血吸虫隔离开来。在这里, 我们描述了一种 SjEV 隔离的替代方法。此方法从 100 nm 到 400 nm, 在成年的日本血吸虫中产生 SjEVs 的宽范围大小。下面的细胞摄取检?...

胞外囊泡 (EVs) 是一种含有多种蛋白质、脂质和核酸的小膜状囊泡的种群。最近的研究表明, 电动车在细胞通讯中起着至关重要的作用, 并参与了许多生理过程的调节, 包括细胞发育、免疫调节、血管生成和细胞迁移2, 3,4,5。累积证据表明, 电动车、循环外来体及其 miRNA 货物代表某些疾病的潜在生物标志物 6.
一些原生动物如阴道毛滴虫、锥虫和利什曼原虫, 已被证明能够分泌电动车;此外, 还发现蠕虫将 EVs 分泌到活主机7中。寄生电动车已被证明涉及感染的维护, 致病性8和免疫调节9。最近在血吸虫的研究中,日本曼氏(曼氏) 10,11和日本血吸虫12已经表明, 成年血吸虫分泌外切样的水泡, 可能是参与特定生物过程的功能规范。
迄今为止, 已有几种方法用于分离胞外囊泡, 如离心、超滤13、聚合物基试剂的使用、大小排除色谱和免疫亲和层析隔离。这些不同的方法具有各自的优点和局限性14。一般认为, 离心是囊泡分离的金标准方法。但是, 此方法受到可能的 EV 聚合14的限制。
在血吸虫中, 已报告了两种 EV 隔离的方法: 包括离心11、12、10和使用商用 EV 隔离套件16等几个阶段 (鸡蛋16,血吸虫童虫11, 成人血吸虫10, 12, 17).鉴于微泡的尺寸范围从数以千纳米到上千奈米, 我们开发了一种替代方法, 结合使用台式离心机, 超滤, 和商用 EV 隔离套件, 以隔离电动车从成年的日本血吸虫。隔离的电动车通常拥有直径 100-400 nm, 并成功地内化的接收细胞。

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		<col />
		<col />
		<col />
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	<tbody>
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			<td height="31" style="height:31px;width:459px;">Material</td>
			<td style="width:196px;"></td>
			<td style="width:188px;"></td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:459px;">Total Exosome Isolation Reagent (from cell culture media)</td>
			<td style="width:196px;">ThermoFisher SCIENTIFIC</td>
			<td style="width:188px;">4478359</td>
			<td style="width:319px;">For isolating S. japonicum extracellular vesicles</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:459px;">PKH67</td>
			<td style="width:196px;">Sigma-aldrich</td>
			<td style="width:188px;">MINI67</td>
			<td style="width:319px;">For labeling Evs</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">RPMI 1640 Medium</td>
			<td style="width:196px;">ThermoFisher SCIENTIFIC</td>
			<td>11875119</td>
			<td>For parasite culture</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Penicillin-Streptomycin</td>
			<td style="width:196px;">ThermoFisher SCIENTIFIC</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">0.22&#956;m syringe filter</td>
			<td style="width:196px;">Merck</td>
			<td>SLGP033RB</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">SnakeSkin Dialysis Tubing</td>
			<td style="width:196px;">Thermo SCIENTIFIC</td>
			<td>68035</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">PEG8000</td>
			<td style="width:196px;">Sangon Biotech</td>
			<td>A100159&#160;</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">RIPA</td>
			<td style="width:196px;">Beyotime</td>
			<td>P0013B</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">EMD Millipore&#8482; Immobilon&#8482; Western Chemiluminescent HRP Substrate (ECL)</td>
			<td style="width:196px;">Fisher scientific</td>
			<td>WBKLS0100&#160;</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">DAPI</td>
			<td>Cell Signaling Technology</td>
			<td>4083</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">BCA kit</td>
			<td style="width:196px;">Beyotime</td>
			<td>P0010</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">CD63 antibodies</td>
			<td style="width:196px;">Sangon Biotech</td>
			<td>D160973-0025</td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">PVDF</td>
			<td>Bio-Rad</td>
			<td>1704273</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Formvar/Carbon 400 mesh</td>
			<td>Ted Pella</td>
			<td>01754-F</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Phosphotungstic Acid</td>
			<td>Ted Pella</td>
			<td>19402</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;">anti-mouse IgG-HRP</td>
			<td>CWBIO</td>
			<td>CW0102</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:459px;">NCTC clone 1469 cells</td>
			<td style="width:196px;">ATCC</td>
			<td style="width:188px;">ATCC&#174; CCL-9.1&#8482;</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:459px;">FBS</td>
			<td style="width:196px;">HyClone</td>
			<td style="width:188px;">SV30087.02</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:459px;">Equipments</td>
			<td style="width:196px;"></td>
			<td style="width:188px;"></td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">GE chemoluminescance imaging system</td>
			<td>GE</td>
			<td>ImageQuant LAS4000mini</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">Transmission electron microscopy</td>
			<td>Hitachi</td>
			<td>H-7600</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Milli-Q water</td>
			<td>Milli-Q</td>
			<td>Milli-Q Elix</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Eppendor Centrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5804R</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">Zetasizer Nano</td>
			<td>Malvern</td>
			<td>Zetasizer Nano ZS&#160;</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Ultracentrifuge</td>
			<td style="width:196px;">Beckman</td>
			<td colspan="2">Optima L-100 XP Ultracentrifuge</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">Incubator</td>
			<td style="width:196px;">ESCO</td>
			<td>CCL-107B</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">Microscope</td>
			<td style="width:196px;">Zeiss</td>
			<td>Zeiss Axin Observer Z1</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and characterization of extracellular vesicles from adult schistosoma japonicum

胞外囊泡 (EVs) 是由多种细胞释放到胞外微环境中的膜状囊泡。EVs 代表了异质泡的种群, 其大小范围介于40和 1000 nm 之间。积累的证据表明, 电动车在病原宿主相互作用中起着重要的调控作用。对血吸虫电动车的深入了解, 应能洞察血吸虫与宿主相互作用的机制, 从而有助于制定防治血吸虫病的新策略。在这里, 我们的目的是进一步研究电动车在血吸虫的功能, 提出了一个协议, 以隔离和特征的电动车从成年的日本血吸虫(S. 血吸虫)。使用离心结合商用外切隔离套件, 从体外培养基中分离出电动汽车。隔离的日本血吸虫EVs (SjEVs) 通常具有 100-400 nm 的直径, 其特征是透射电镜和西方印迹。PKH67 染料标记 SjEVs 的使用表明, SjEVs 是由受体细胞内部化的。总的来说, 我们的协议提供了一种从成人血吸虫隔离电动车的替代方法;隔离 SjEVs 可适用于功能分析。

为了用所描述的协议来量化孤立 SjEVs 的产量, 我们利用 SjEVs 蛋白质检测方法, 从28天成年血吸虫中获取分离的蛋白质浓度。如表 1所示, SjEV 蛋白浓度从208µg 到250µg 每100毫升培养基 (表 1) 不等。由马尔文纳米粒子分析确定的粒子大小表明, 隔离的 SjEVs 从 100 nm 到 400 nm 不等, 在大小上大约 220 nm (图 1)。通过透射电镜对 SjEVs 的进?...

Watch this Scientific Journal Video about Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum at JoVE.com

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

成年日本血吸虫细胞外囊泡的分离与鉴定

在这里, 我们提出一个协议, 以描述细胞外囊泡 (电动车) 隔离在体外培养培养基从成年日本血吸虫。

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

Research

JoVE Journal

Immunology and Infection

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Cercarial转型和在体外栽培曼氏血吸虫 Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

科研

免疫与感染

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

从使用鸡蛋<em&gt;血吸虫</em&gt;作为<em&gt;在体内</em&gt;蠕虫引起的肺部炎症模型

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

从老鼠肠道树突状细胞和巨噬细胞的分离和表征

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

简化人力中性粒细胞胞外陷阱（英语教师）分离和处理

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

隔离和中性粒细胞的表征与抗肿瘤性质

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

分离，鉴定和牙龈免疫细胞网络的功能性考试

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

小鼠小肠淋巴细胞的分离和流式细胞表征

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Flow Virometry to Analyze Antigenic Spectra of Virions and Extracellular Vesicles

流量Virometry来分析病毒颗粒的抗原谱和外囊泡

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of ...

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

Legionella pneumophila Outer Membrane Vesicles: Isolation and Analysis of Their Pro-inflammatory Potential on Macrophages

嗜肺军团菌外膜囊泡：隔离及对巨噬细胞促炎潜力分析

Bacteria are able to secrete a variety of molecules via various secretory systems. Besides the secretion of molecules into the extracellular space or ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

受肥胖相关炎症组织的巨噬细胞的分离、鉴定和纯化

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...