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

Denne studien var delvis eller helt, støttes av National Natural Science Foundation i Kina (31472187 og 31672550) og landbruksvitenskap og teknologi innovasjon Program for det kinesiske akademi for landbruksvitenskap.

Alle dyreforsøk ble godkjent av den lokale etikk av Shanghai Veterinary Research Institute, kinesiske akademi for landbruksvitenskap (tillater tall: SHVRIAU-14-0101).
1. i Vitro kulturen i Schistosomes
Merk: Schistosomes representerer en biohazard. Arbeidere bør ha gummihansker på alle tider når håndtering av ormer, schistosomal suspensjon eller andre relaterte biologisk materiale. New Zealand kaniner var infisert med ~...

<ol>
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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+Extracellular+Vesicles:+Biogenesis,+RNA+Cargo+Selection,+Content,+Release,+and+Uptake.">Introduction to Extracellular Vesicles: Biogenesis, RNA Cargo Selection, Content, Release, and Uptake.</a> Cell Mol Neurobiol. 36 (3), 301-312 (2016).</li><li>Riaz, F., Cheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome-like+vesicles+of+helminths:+implication+of+pathogenesis+and+vaccine+development.">Exosome-like vesicles of helminths: implication of pathogenesis and vaccine development.</a> Ann Transl Med. 5 (7), 175 (2017).</li><li>Zhang, H. G., Grizzle, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+and+cancer:+a+newly+described+pathway+of+immune+suppression.">Exosomes and cancer: a newly described pathway of immune suppression.</a> Clin Cancer Res. 17 (5), 959-964 (2011).</li><li>Colombo, M., Raposo, G., Thery, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis,+secretion,+and+intercellular+interactions+of+exosomes+and+other+extracellular+vesicles.">Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.</a> Annu Rev Cell Dev Biol. 30, 255-289 (2014).</li><li>Li, 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=Circular+RNA+is+enriched+and+stable+in+exosomes:+a+promising+biomarker+for+cancer+diagnosis.">Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis.</a> Cell Res. 25 (8), 981-984 (2015).</li><li>Marcilla, 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=Extracellular+vesicles+in+parasitic+diseases.">Extracellular vesicles in parasitic diseases.</a> J Extracell Vesicles. 3, 25040 (2014).</li><li>Cwiklinski, K., 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=The+Extracellular+Vesicles+of+the+Helminth+Pathogen,+Fasciola+hepatica:+Biogenesis+Pathways+and+Cargo+Molecules+Involved+in+Parasite+Pathogenesis.">The Extracellular Vesicles of the Helminth Pathogen, Fasciola hepatica: Biogenesis Pathways and Cargo Molecules Involved in Parasite Pathogenesis.</a> Mol Cell Proteomics. 14 (12), 3258-3273 (2015).</li><li>Buck, A. H., 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=Exosomes+secreted+by+nematode+parasites+transfer+small+RNAs+to+mammalian+cells+and+modulate+innate+immunity.">Exosomes secreted by nematode parasites transfer small RNAs to mammalian cells and modulate innate immunity.</a> Nat Commun. 5, 5488 (2014).</li><li>Sotillo, 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+secreted+by+Schistosoma+mansoni+contain+protein+vaccine+candidates.">Extracellular vesicles secreted by Schistosoma mansoni contain protein vaccine candidates.</a> Int J Parasitol. 46 (1), 1-5 (2016).</li><li>Nowacki, F. C., 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=Protein+and+small+non-coding+RNA-enriched+extracellular+vesicles+are+released+by+the+pathogenic+blood+fluke+Schistosoma+mansoni.">Protein and small non-coding RNA-enriched extracellular vesicles are released by the pathogenic blood fluke Schistosoma mansoni.</a> J Extracell Vesicles. 4, 28665 (2015).</li><li>Wang, L., 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=Exosome-like+vesicles+derived+by+Schistosoma+japonicum+adult+worms+mediates+M1+type+immune-+activity+of+macrophage.">Exosome-like vesicles derived by Schistosoma japonicum adult worms mediates M1 type immune- activity of macrophage.</a> Parasitol Res. 114 (5), 1865-1873 (2015).</li><li>Koh, Y. Q., Almughlliq, F. B., Vaswani, K., Peiris, H. N., Mitchell, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome+enrichment+by+ultracentrifugation+and+size+exclusion+chromatography.">Exosome enrichment by ultracentrifugation and size exclusion chromatography.</a> Front Biosci (Landmark Ed). 23, 865-874 (2018).</li><li>Safdar, A., Saleem, A., Tarnopolsky, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+endurance+exercise-derived+exosomes+to+treat+metabolic+diseases.">The potential of endurance exercise-derived exosomes to treat metabolic diseases.</a> Nat Rev Endocrinol. 12 (9), 504-517 (2016).</li><li>Livshits, M. 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=Isolation+of+exosomes+by+differential+centrifugation:+Theoretical+analysis+of+a+commonly+used+protocol.">Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol.</a> Sci Rep. 5, 17319 (2015).</li><li>Zhu, 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=Release+of+extracellular+vesicles+containing+small+RNAs+from+the+eggs+of+Schistosoma+japonicum.">Release of extracellular vesicles containing small RNAs from the eggs of Schistosoma japonicum.</a> Parasit Vectors. 9 (1), 574 (2016).</li><li>Zhu, L., 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=Molecular+characterization+of+S.+japonicum+exosome-like+vesicles+reveals+their+regulatory+roles+in+parasite-host+interactions.">Molecular characterization of S. japonicum exosome-like vesicles reveals their regulatory roles in parasite-host interactions.</a> Sci Rep. 6, 25885 (2016).</li><li>Lewis, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> Curr Protoc Immunol. , 11 (2001).</li><li>Cheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+miRNAs:+roles+in+cancer+diagnosis,+prognosis+and+therapy.">Circulating miRNAs: roles in cancer diagnosis, prognosis and therapy.</a> Adv Drug Deliv Rev. 81, 75-93 (2015).</li></ol>

Nyere studier på EVs har vist at schistosome EVs spiller en viktig rolle i vert-patogen interaksjoner3,9,12,16. Til ytterligere adressen deres juridiske funksjoner, er det viktig å isolere EVs fra schistosomes. Her beskriver vi en alternativ metode for SjEV isolasjon. Denne metoden gir en rekke størrelsen på SjEVs, 100 nm til 400 nm, i voksen S. japonicum. De følgende cellen oppta...

Ekstracellulære blemmer (EVs) er en befolkning på små membran-bundet blemmer innkapslet med ulike proteiner, lipider og atomer syren. Nyere studier har vist at EVs spille en avgjørende rolle i celle-celle kommunikasjon, og er involvert i regulering av mange fysiologiske prosesser, inkludert celle utvikling, immun regulering, angiogenese og celle migrasjon2, 3,4,5. Samler bevis indikerer at EVs, sirkulerer exosomes og frakten miRNA representerer potensielle biomarkers av visse sykdommer6.
Flere protozoer som Trichomonas vaginalis, Trypanosoma cruziog Leishmania spp., har vist seg å bli kjøpedyktig skille ut EVs; helminths i tillegg er funnet å skille ut EVs til levende vert7. Parasittiske EVs har vist seg å være involvert i vedlikehold av infeksjon, virusets8og immun regulering9. Nyere studier i schistosomes begge Schistosoma mansoni (S. mansoni) 10,11 og S. japonicum12 har indikert at voksen schistosomes skiller exosome som blemmer som kan involvert i funksjonelle forskrift av bestemte biologiske prosesser.
Hittil har flere metoder brukt isolere ekstracellulære blemmer, som ultracentrifugation, ultrafiltrasjon13, bruk av polymer-baserte reagenser, størrelse-utestenging kromatografi og immunoaffinity isolasjon. Disse forskjellige metodene har sine egne fordeler og begrensninger14. Ultracentrifugation regnes vanligvis metoden gullstandarden for vesicle isolasjon. Men lider denne metoden av begrensning av potensielle EV aggregering14.
I schistosomes, et par metoder er rapportert for EV isolasjon: ultracentrifugation11,12,10 og bruk av kommersielle EV isolering kit16 for flere etapper (egg16, schistosomula11, voksen schistosomes10,12,17). Gitt at microvesicles er av en rekke størrelse fra flere hundre nanometer tusen nanometer, utviklet vi en alternativ metode som kombinerer med bruk av benken sentrifuge, ultrafiltrasjon og en kommersiell EV isolering kit å isolere EVs fra voksen Schistosoma japonicum. De isolerte EVs vanligvis hadde en diameter på 100-400 nm og var vellykket internalisert mottaker cellene.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="31">
			<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

Ekstracellulære blemmer (EVs) er membranous blemmer utgitt av en rekke celler i den ekstracellulære microenvironment. EVs representerer innbyggere heterogene blemmer, hvis størrelse varierer mellom 40 og 1000 nm. Akkumulerte bevis indikerte at EVs spille viktige forskrifter roller i patogen vert interaksjoner. En dyp forståelse av schistosome EVs skal gi innsikt i mekanismene bak schistosome vert interaksjoner, slik at utviklingen av romanen strategier mot schistosomiasis. Her, mål vårt er å ytterligere studere EVs funksjoner i schistosomes ved å presentere en protokoll for isolering og karakterisering av EVs fra voksen Schistosoma japonicum (S. japonicum). EVs ble isolert fra i vitro kultur medium med sentrifugering kombinert med en kommersiell exosome isolering kit. Den isolerte S. japonicum EVs (SjEVs) vanligvis har en diameter på 100-400 nm, og er preget av overføring elektronisk mikroskopi og vestlige blotting. Bruken av PKH67 dye-merket SjEVs har vist at SjEVs er internalisert mottaker cellene. Samlet gir våre protokollen en alternativ metode for å isolere EVs fra voksen schistosomes; den isolerte SjEVs kan være egnet for funksjonsanalyse.

For å kvantifisere avkastningen av isolerte SjEVs bruker beskrevet protokollen, brukte vi BCA protein analysen til protein konsentrasjonen av den isolerte SjEVs fra 28-dagers voksen schistosomes. Som vist i tabell 1, varierte SjEV protein konsentrasjonen 208 µg til 250 µg per 100 mL medium (tabell 1). Partikkelstørrelse, som Malvern hydrogenion analyse, indikerte at den isolerte SjEVs varierte fra 100 nm til 400 nm, med høyest befolkning rundt 220 nm...

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

Isolering og karakterisering av ekstracellulære blemmer fra voksen Schistosoma japonicum

Her presenterer vi en protokoll for å beskrive ekstracellulære blemmer (EVs) isolasjon i vitro kulturperler medium fra voksen Schistosoma japonicum.

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

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

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

Bruke Egg fra<em&gt; Schistosoma mansoni</em&gt; Som en<em&gt; In vivo</em&gt; Modell av Helminth-indusert lungebetennelse

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

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Isolering og karakterisering av dendrittiske celler og makrofager fra musen Intestine

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

Forenklede menneske Nøytrofile Ekstracellulære Traps (NET) Isolasjon og håndtering

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Isolering og karakterisering av nøytrofiler med anti-tumor egenskaper

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

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Isolasjon, karakterisering og Funksjonell Undersøkelse av Gingival immun Cell Network

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

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Isolasjon og Flowcytometriske Karakterisering av Murine Small Intestinal Lymfocytter

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

Flow Virometry å analysere Antigen Spectra av Virioner og ekstracellulære vesikler

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

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

Legionella pneumophila ytre membran Blemmer: Isolering og analyse av deres Pro-inflammatorisk potensialet på Makrofager

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