Name: evaluation of extracellular vesicle function during malaria infection
Uploaded: 2018-02-14T14:30:00
Description: Watch this Scientific Journal Video about Evaluation of Extracellular Vesicle Function During Malaria Infection at JoVE.com

这项研究由诺华医学和生物研究基金会 (PYM)、戈特弗里德和朱莉娅 Bangerter Rhyner 基金会 (兆瓦和 PYM) 和大学 (PYM) 的研究池等部分财政支助。额外赠款包括瑞士政府优秀奖学金为外国学者 (对 KAB 和 SM)。我们感谢伊莎贝尔 Fellay 和 Solange Kharoubi 赫斯的技术支持。

根据 Swissethics (swissethics.ch) 的指导原则, 从健康捐赠者的血液中获得人体红细胞。
注意:恶性疟原虫寄生虫培养 (3D7) 和 EV 生产以前是在 Mbagwu ( et al) 中描述的。11由于P. 恶性疟原虫是人类病原体, 请查阅当地的规章以进行处理。文化应该保持不育的整个时间。
1. 电动汽车的荧光标记
注: 以下程序利?...

<ol>
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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=Extracellular+Vesicles+from+Trypanosoma+brucei+Mediate+Virulence+Factor+Transfer+and+Cause+Host+Anemia.">Extracellular Vesicles from Trypanosoma brucei Mediate Virulence Factor Transfer and Cause Host Anemia.</a> Cell. 164 (1-2), 246-257 (2016).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+genomics,+biogenesis,+mechanism,+and+function.">MicroRNAs: genomics, biogenesis, mechanism, and function.</a> Cell. 116 (2), 281-297 (2004).</li><li>Mantel, P. 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=Infected+erythrocyte-derived+extracellular+vesicles+alter+vascular+function+via+regulatory+Ago2-miRNA+complexes+in+malaria.">Infected erythrocyte-derived extracellular vesicles alter vascular function via regulatory Ago2-miRNA complexes in malaria.</a> Nat Commun. 7, 12727 (2016).</li><li>Mbagwu, S., Walch, M., Filgueira, L., Mantel, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+Characterization+of+Extracellular+Vesicles+in+Malaria.">Production and Characterization of Extracellular Vesicles in Malaria.</a> Methods Mol Biol. 1660, 377-388 (2017).</li><li>Schweitzer, K. M., 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=Characterization+of+a+newly+established+human+bone+marrow+endothelial+cell+line:+distinct+adhesive+properties+for+hematopoietic+progenitors+compared+with+human+umbilical+vein+endothelial+cells.">Characterization of a newly established human bone marrow endothelial cell line: distinct adhesive properties for hematopoietic progenitors compared with human umbilical vein endothelial cells.</a> Lab Invest. 76 (1), 25-36 (1997).</li><li>Wong, W., Farr, R., Joglekar, M., Januszewski, A., Hardikar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probe-based+Real-time+PCR+Approaches+for+Quantitative+Measurement+of+microRNAs.">Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs.</a> J Vis Exp. (98), (2015).</li><li>Mulcahy, L. A., Pink, R. C., Carter, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routes+and+mechanisms+of+extracellular+vesicle+uptake.">Routes and mechanisms of extracellular vesicle uptake.</a> J Extracell Vesicles. 3, (2014).</li><li>French, K. C., Antonyak, M. A., Cerione, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicle+docking+at+the+cellular+port:+Extracellular+vesicle+binding+and+uptake.">Extracellular vesicle docking at the cellular port: Extracellular vesicle binding and uptake.</a> Semin Cell Dev Biol. 67, 48-55 (2017).</li><li>Montecalvo, A., 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=Mechanism+of+transfer+of+functional+microRNAs+between+mouse+dendritic+cells+via+exosomes.">Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes.</a> Blood. 119 (3), 756-766 (2012).</li><li>Idro, R., Jenkins, N. E., Newton, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis,+clinical+features,+and+neurological+outcome+of+cerebral+malaria.">Pathogenesis, clinical features, and neurological outcome of cerebral malaria.</a> Lancet Neurol. 4 (12), 827-840 (2005).</li><li>Jambou, R., 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=Plasmodium+falciparum+adhesion+on+human+brain+microvascular+endothelial+cells+involves+transmigration-like+cup+formation+and+induces+opening+of+intercellular+junctions.">Plasmodium falciparum adhesion on human brain microvascular endothelial cells involves transmigration-like cup formation and induces opening of intercellular junctions.</a> PLoS Pathog. 6 (7), e1001021 (2010).</li><li>Zhou, 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=Cancer-secreted+miR-105+destroys+vascular+endothelial+barriers+to+promote+metastasis.">Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis.</a> Cancer Cell. 25 (4), 501-515 (2014).</li></ol>

一些寄生虫, 包括弓形虫、锥、利什曼原虫和滴虫, 触发受感染的宿主细胞释放电动车。根据病原体, 释放的电动车可以调节宿主免疫应答或调解寄生虫之间的细胞通讯6。然而, 几乎没有证据表明这些小囊泡是如何导致疟疾疾病的。在这里, 我们描述了几种方法来调查在疟原虫感染期间 EVs 的功能。例如, 通过用 PKH67 荧光染料对其进行标记, 可以有效地监测接收单元体内</e...

根据世界卫生组织的报告, 2015年全世界有2亿1200万起新的疟疾病例, 大约42.9万人死亡, 主要是五岁以下的儿童1。导致严重疾病的机制, 通常与血管功能障碍有关, 仍未定义为2。疟原虫-iRBCs 分泌小的双脂膜球体, 称为胞外囊泡 (EVs)。众所周知, 这些电动车与感染过程和宿主对感染的免疫反应有潜在的相关性;然而, 对这些小泡在疟疾感染期间的确切功能知之甚少3。他们有可能扮演两个重要的角色: 一方面, 他们可能通过激活巨噬细胞4,5来促进发病机制;另一方面, 他们可能会调解寄生虫之间的细胞通讯和寄生虫和主机6,7。事实上, 寄生虫可以通过电动车相互转移蛋白质或核酸。例如,锥寄生虫罗得西亚 EVs 可以转移毒力因子血清抗性相关 (), 并可针对其他 T . 寄生虫和宿主红细胞 8.此外,恶性疟原虫-iRBCs 通过在电动车内转移核酸来相互通信。这使得寄生虫能够优化和同步其生长。实际上, EVs 可能是 gametocyte 转换的主要调节器, 因此有助于对传输阶段7的调节。
电动车不仅能调节寄生虫, 而且还能调解寄生宿主之间的相互作用。我们最近发现, 电动车从 iRBCs 包含主机衍生的 microRNAs (miRNAs; 小 RNA 物种的范围内21-25 核苷酸9), 由人类内皮细胞占去。在电动车中的 miRNAs 形成一个稳定的复杂与 Ago2 (RNA 诱导沉默复合体的成员), 一旦交付给接受细胞, 能够明确地沉默基因表达和影响细胞的屏障属性10。为研究电动汽车的功能, 开发了标准协议。在这里, 我们首先描述一个协议, 允许电动车的荧光标记, 以调查他们的接收细胞的吸收。此外, 通过使用共焦显微镜, 可以跟踪 EV 的命运在细胞内。几种荧光染料可用于跟踪电动车。胺反应染料, 5 (和-6)-羧基双乙酸琥珀酸酯 (CFSE) 和 Calcein-在囊泡内成为荧光。我们更喜欢使用两亲的标签, PKH, 因为它给一个更明亮和更均匀的信号。此方法为了解 EVs 和接收单元之间的交互提供了重要信息。在某些情况下, 电动车绑定到细胞的表面, 一些囊泡迅速占去。在吸收后, 电动车将他们的货物运送到细胞中, 并在其中发挥调节作用。
在这里, 我们描述了一个协议, 以测量内皮细胞的屏障功能在体外通过量化的转移荧光葡聚糖通过细胞单层。更灵敏的示踪剂可以使用, 如核素标记。但是, 它们需要特殊的安全预防措施才能使用。还有其他的化验方法来测量体外屏障功能, 如 transendothelial 电阻 (志愿者), 它测量紧密接合的完整性。最后可视化 ZO-1, 一个紧密的连接蛋白, 通过免疫荧光允许评估紧密连接完整性以及10。由于 EVs 是复杂和异构实体, 包含若干具有潜在监管特性的货物, 因此过度表达 tshr 一个特定的 RNA 来研究它对接收单元的影响是有用的。因此, 我们还定义了一个协议, 其目的是生成稳定的单元格线, 表达感兴趣的 miRNA10。

<table>
	<tbody>
		<tr>
			<td>PKH67 Green Fluorescent Cell Linker Mini Kit</td>
			<td>Sigma-Aldrich</td>
			<td>MINI67-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Diluent C</td>
			<td>Sigma-Aldrich</td>
			<td>G8278</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P8920</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFisher - Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Phalloidin CF594</td>
			<td>Biotium</td>
			<td>#00045</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>ThermoFisher</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFisher</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine B isothiocyanate&#8211;Dextran</td>
			<td>ThermoFisher</td>
			<td>R9379-250MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Insert with PET membrane transparent Falcon&#160;for plate 24 wells</td>
			<td>Falcon</td>
			<td>353095</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth&#160;Medium MV</td>
			<td>Promocell</td>
			<td>C-22020</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9620-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>MTS Cell Proliferation Colorimetric Assay Kit</td>
			<td>Biovision</td>
			<td>K300-500</td>
			<td></td>
		</tr>
		<tr>
			<td>hexadimethrine bromide</td>
			<td>Sigma-Aldrich</td>
			<td>107689-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>MISSION&#160;Lenti microRNA, Human hsa-miR-451a</td>
			<td>Sigma-Aldrich</td>
			<td>HLMIR0583</td>
			<td></td>
		</tr>
		<tr>
			<td>MISSION Lenti microRNA, ath-miR416, Negative Control 1 Transduction Particles</td>
			<td>Sigma-Aldrich</td>
			<td>NCLMIR001</td>
			<td></td>
		</tr>
		<tr>
			<td>MISSION&#160;Lenti microRNA, Human</td>
			<td>Sigma-Aldrich</td>
			<td>NCLMIR0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica TCS SP5</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy mini Kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan MicroRNA Reverse Transcription Kit 1000 reactions</td>
			<td>ThermoFisher</td>
			<td>4366597</td>
			<td></td>
		</tr>
		<tr>
			<td>hsa-mir-451a&#160;RT/750 PCR rxns</td>
			<td>ThermoFisher</td>
			<td>001141</td>
			<td></td>
		</tr>
		<tr>
			<td>U6 snRNA</td>
			<td>ThermoFisher</td>
			<td>001973</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Universal Master Mix II, with UNG</td>
			<td>ThermoFisher</td>
			<td>4440038</td>
			<td></td>
		</tr>
		<tr>
			<td>StepOnePlus Real-Time PCR System</td>
			<td>ThermoFisher</td>
			<td>4376600</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of extracellular vesicle function during malaria infection

疟疾是由疟原虫寄生虫引起的危及生命的疾病, 而P. 恶性疟原虫是非洲大陆上最普遍的, 对全球大多数与疟疾有关的死亡负责。包括寄生虫在组织中的封存、血管功能障碍和炎症反应等几个因素影响了疟疾感染者的病情演变。恶性疟原虫-感染的红细胞 (iRBCs) 释放出小的胞外囊泡 (EVs), 其中含有不同种类的货物分子, 它介导寄生虫与宿主之间的致病机制和细胞间通讯。电动汽车是由它们调节其功能的细胞有效地占用的。在这里, 我们讨论的策略, 以解决电动车在寄生虫宿主互动的作用。首先, 我们描述了一种直接的方法来标记和跟踪的 EV 内化的内皮细胞, 使用绿色细胞链接染料。第二, 我们报告了一个简单的方法来测量渗透率跨内皮细胞单层, 使用荧光标记葡聚糖。最后, 我们展示了如何研究小的非编码 RNA 分子在内皮细胞功能中的作用。

在这里, 我们描述了研究电动汽车与宿主细胞相互作用的协议。荧光标记的 EVs 的摄取是通过共焦显微镜 (图 1) 来监测的。内皮细胞有效地占去了电动车, 但是利用 evs 的潜伏期可以优化以跟踪吸收。为了更好地定位电动汽车内部的细胞, 染色肌动蛋白与罗丹明。接下来, 我们使用一个过滤膜的顶端, 其中一层内皮细胞生长。罗丹明 B 异硫氰酸酯-葡聚糖?...

Watch this Scientific Journal Video about Evaluation of Extracellular Vesicle Function During Malaria Infection at JoVE.com

Evaluation of Extracellular Vesicle Function During Malaria Infection

在这项工作中, 我们描述了研究由恶性疟原虫感染的红细胞释放出的细胞外囊 (EVs) 作用的协议。特别是, 我们关注的是电动车与内皮细胞的相互作用。

疟疾感染中胞外囊泡功能的评价

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

Research

JoVE Journal

Immunology and Infection

Neutrophil Extracellular Traps: How to Generate and Visualize Them

嗜中性粒细胞胞外的陷阱：如何生成和可视化

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them ...

科研

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A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

一个简单的的Chelex协议对DNA的提取<em&gt;按蚊</em&gt;属。

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

2D and 3D Chromosome Painting in Malaria Mosquitoes

疟疾蚊子中的二维和三维染色体绘画

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization

Antecubital 静脉插管高强度运动时血乳酸和血浆胰岛素的评价

The measurement of metabolic and endocrinal markers during physical activity is of relevance to understanding the physiological implications of different ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

成像 Ca2 +在志贺菌感染上皮细胞期间的响应

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ ...

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

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Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature ...

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

使用 NETQUANT 对中性粒细胞外陷阱的自动基于图像的定量

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

高产细胞外囊泡制剂的纯化远离病毒

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...