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 undersøgelse var delvist eller helt, støttet af National Natural Science Foundation of China (31472187 og 31672550) og landbruget videnskab og teknologi Innovation Program af det kinesiske Musikkonservatorium Jordbrugsforskning.

Alle dyreforsøg blev godkendt af det lokale etiske udvalg af Shanghai veterinære forskningsinstitut, kinesiske akademi for Jordbrugsforskning (tillade nummer: SHVRIAU-14-0101).
1. in Vitro kultur af Schistosomes
Bemærk: Schistosomes repræsenterer en biohazard. Arbejdstagere skal bære latex handsker på alle tidspunkter, når håndtering af orme, schistosomal suspensioner, eller andre relaterede biologiske materialer. Ne...

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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. 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De seneste undersøgelser af evt har demonstreret at schistosome evt spiller en vigtig rolle i vært-patogen interaktioner3,9,12,16. Til yderligere adresse deres regulerende funktioner, er det vigtigt at isolere evt fra schistosomes. Her beskriver vi en alternativ metode til SjEV isolation. Denne metode giver en bred vifte af SjEVs, størrelse fra 100 nm til 400 nm, i voksen S. japonicum</e...

Ekstracellulære vesikler (EVs) er en befolkning på lille membran-bundet vesikler indkapslet med forskellige proteiner, lipider og nukleinsyrer. Nylige undersøgelser viste at evt spiller en afgørende rolle i celle-celle kommunikation, og er involveret i reguleringen af mange fysiologiske processer, herunder celle udvikling, immun forordning, angiogenese og celle migration2, 3,4,5. Akkumulere beviser indikerer at evt, cirkulerende exosomes, og deres miRNA last repræsenterer potentielle biomarkører for visse sygdomme6.
Flere protozoer såsom Trichomonas vaginalis, Trypanosoma cruziog Leishmania spp., har vist sig at være i stand til at udskille evt; helminter har desuden fundet at udskille evt i levende vært for7. Parasitære evt har vist sig at være deltager i vedligeholdelse af infektion, patogenicitet8og immun forordning9. Nylige undersøgelser i schistosomes både Schistosoma mansoni (S. mansoni) 10,11 og S. japonicum12 har tilkendegivet, at voksen schistosomes udskiller exosome-lignende blærer, der kan være involveret i de funktionelle bestemmelser i specifikke biologiske processer.
Til dato, er flere metoder blevet brugt til at isolere ekstracellulære vesikler, såsom ultracentrifugering, ultrafiltrering13, brugen af polymer-baserede reagenser, størrelse-udelukkelse kromatografi og immunoaffinity isolation. Disse forskellige metoder har deres egne fordele og begrænsninger14. Generelt anses ultracentrifugering metoden guldstandarden for vesikel isolation. Imidlertid lider denne metode begrænsning af potentielle EV sammenlægning14.
I schistosomes, et par metoder er blevet rapporteret til EV isolation: disse omfatter ultracentrifugering11,12,10 og anvendelse af kommercielle EV isolering kit16 til flere faser (æg16, schistosomula11, voksen schistosomes10,12,17). Microvesicles er af en bred vifte af størrelse fra flere hundrede nanometer til tusinde nanometer, udviklet vi en alternativ metode, der kombinerer med brug af bænken centrifuge, ultrafiltrering og en kommerciel EV isolering kit at isolere evt fra voksen Schistosoma japonicum. De isolerede evt typisk havde en diameter på 100-400 nm og blev med held internaliseret af de modtagende celler.

<table border="0" cellpadding="0" cellspacing="0" style="width:1161px;" width="1160">
	<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 vesikler (EVs) er hindeagtige vesikler udgivet af en række celler i den ekstracellulære mikromiljø. Evt repræsenterer en befolkning af heterogene vesikler, hvis størrelse varierer mellem 40 og 1000 nm. Akkumulerede beviser angivet at evt spiller en vigtig lovgivningsmæssig rolle i pathogen-værtssammenspil. En dyb forståelse af schistosome evt skal give indsigt i de underliggende schistosome-værtssammenspil, muliggør udviklingen af nye strategier mod Bilharziose mekanismer. Her, vi sigter mod at yderligere undersøgelse evt-funktioner i schistosomes ved at præsentere en protokol til isolering og karakterisering af evt fra voksen Schistosoma japonicum (S. japonicum). Evt blev isoleret fra in vitro- dyrkningsmediet ved hjælp af centrifugering kombineret med en kommerciel exosome isolering kit. Den isolerede S. japonicum evt (SjEVs) typisk har en diameter på 100-400 nm, og er karakteriseret ved transmission elektroniske mikroskopi og western blotting. Brugen af PKH67 dye-mærket SjEVs har vist, at SjEVs er internaliseret af de modtagende celler. Samlet set giver vores protokol en alternativ metode til at isolere evt fra voksen schistosomes; den isolerede SjEVs kan være egnet til funktionelle analyse.

For at kvantificere udbyttet af isolerede SjEVs ved hjælp af den beskrevne protokol, brugte vi BCA protein assay adgang til proteinkoncentration af isolerede SjEVs fra 28-dages voksne schistosomes. Som vist i tabel 1, SjEV proteinkoncentration varierede fra 208 µg til 250 µg pr. 100 mL af medium (tabel 1). Partikelstørrelse, som bestemmes af Malvern nanopartikel analyse, angivet, at den isolerede SjEVs varierede fra 100 nm til 400 nm, med den højeste...

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 af ekstracellulære vesikler fra voksen Schistosoma japonicum

Vi præsenterer her, en protokol for at beskrive ekstracellulære vesikler (EVs) isolation i in 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

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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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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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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 ...

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Flow Virometry at analysere Antigen Spectra af Virioner og ekstracellulære vesikler

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