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

Denne forskning blev støttet af Li Ka Shing Foundation, Guangdong High Level University Project &quot;Grønne Technologies for Marine Industries&quot;, Natural Sciences Foundation of China (http://www.nsfc.gov.cn/ Grant No. 30971665, 81172894, 81370925) og Education Department of Guangdong (http://www.gdhed.edu.cn/ Grant No.cxzd1123).

8 til 12 ugentlige BALB / c-mus blev købt fra Shanghai Experimental Animal Center (Shanghai, Kina) og opdrættet i et specifikt patogenfrit og luftkonditioneret dyreanlæg. Dyrepleje og forsøgsprocedurer var i overensstemmelse med retningslinjerne for brug og pleje af forsøgsdyr etableret af Shantou University. 1. Montering af apparatet <ol><li> For at sikre sterilitet skal du tænde UV-lyset af en vævskulturhætte i 30 minutter før brug. </li><li> Skru en engangs 25 mm filterenhed ne...

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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> En stor mængde indsats er blevet brugt til at etablere tilgange til gen-, enzym- og celleudskiftningsterapier. Dette har resulteret i store gennembrud og endda kliniske anvendelser 1 , 2 , 3 . For nylig blev en kontroversiel mitokondrierepoterterapi baseret på nukleoverførselsteknologi anvendt til in vitro befrugtning til kvinder i alderdommen eller med en dødelig mitokondriell DNA-mutation 4 . Defekter, der findes i aldersrelaterede sygdomme, herunder aterosklerose, type 2 diabetes, Alzheimers sygdom og kræft, er som regel mange facetter. Det er blevet dokumenteret, at akkumuleringen af ​​lipiddråber; Aflejringen af ​​amyloidprotein; Opbevaring af udfoldede proteiner i det endoplasmatiske retikulum; Og defekt proteasom, autophagosom og mitokondrier bidrager til udviklingen eller forværringen af ​​disse sygdomme&quot;Xref&quot;&gt; 5 , 6 , 7 , 8 , 9 , 10 , 11 . I øjeblikket er der ingen tilgængelig mekanisme rettet mod direkte afhjælpning af funktionsfejl i cytosol og organeller, hvilket forårsager senescens og aldrende fænotyper. Vi har tidligere rapporteret om dannelsen af ​​plasmamembresvesikler (PMV&#39;er) gennem den mekaniske ekstrudering af pattedyrceller 12 . Med undtagelse af kernen blev komponenter i membranen eller cytosolen, herunder proteiner og RNA, såvel som organellerne, såsom mitokondrier, fundet i PMV&#39;er. I det væsentlige kan en PMV betragtes som en miniature enucleeret celle. Endnu vigtigere fik fusion af PMV&#39;er med mitokondrier-defekte Rho0-celler genoprettet mitotisk aktivitet under normale dyrkningsbetingelser. Dette er den første rapport om establiUdvise en potentielt effektiv tilgang til cytoplasm-erstatningsterapi. Knoglemarvs mesenkymale stamceller (BMSC&#39;er) er multipotente stamceller, som rutinemæssigt genereres fra knoglemarven og ekspanderes let i kultur. Embryonale stamcelle markører Oct4, Nanog og SOX2 er blevet påvist ved lave niveauer i MSCs 13 . Telomeraseaktivitet er også målelig. Desuden gør fraværet af co-stimulerende molekyler og humane leukocytantigen (HLA) klasse II-molekyler såvel som lav HLA klasse I-ekspression på MSC&#39;er dem ideelle celler til allogen eller &quot;off-the-shelf&quot; -brug i Både regenerativ medicin og immunmodulerende anvendelser 14 . Her beskriver vi hvordan man forbereder PMV&#39;er fra mus BMSC&#39;er via ekstrudering gennem en polycarbonatmembran med 3 μm porer, bestemmer forekomsten af ​​mitokondrier og undersøge vedligeholdelsen af ​​membranpotentialet i PMV&#39;er ved anvendelse af confocAl mikroskopi, forberede koncentrerede, men ikke aggregerede PMV&#39;er ved centrifugering og udføre in vivo injektionen af ​​PMV&#39;er i muscus gastrocnemius muskelen.

<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

Vi har tidligere rapporteret om dannelsen af ​​plasmamembresvesikler (PMV&#39;er) gennem mekanisk ekstrudering af pattedyrceller. Fusionen af ​​PMV&#39;er med mitochondriale mangelfulde Rho0-celler genoprettede mitotisk aktivitet under normale dyrkningsbetingelser. Aterosklerose, type 2 diabetes, Alzheimers sygdom og kræft er aldersrelaterede sygdomme, der er rapporteret at være forbundet med flere mekaniske og funktionelle defekter i cytosol og organeller af en række celletyper. Knoglemarv mesenkymale stamceller (BMSC&#39;er) repræsenterer en unik cellepopulation fra knoglemarven, som har selvfornyelsesevne, samtidig med at deres multipotensitet opretholdes. Suppleringen af ​​senescensceller med ung cytoplasma fra autologe BMSC&#39;er via fusion af PMV&#39;er giver en lovende tilgang til forbedring eller endog omvendt aldersrelaterede fænotyper. Denne protokol beskriver, hvordan man forbereder PMV&#39;er fra BMSC&#39;er via ekstrudering gennem en polycarbonatmembran med 31; m porer, bestemme forekomsten af ​​mitokondrier og undersøge vedligeholdelsen af ​​membranpotentialet inden for PMV&#39;er ved anvendelse af et konfokalt mikroskop, koncentrere PMV&#39;er ved centrifugering og udføre in vivo injektionen af ​​PMV&#39;er i muskels gastrocnemius muskel.

Nøglen til en vellykket forberedelse af PMV&#39;er afhænger meget af den korrekte samling af filterenheden ( Figur 1 ), som kan testes ved at skubbe 1 ml PBS gennem membranen. Hvis der opstår lækage, skal filterenheden monteres igen og testes igen. Lækage kan dog kun testes pålideligt, når cellerne skubbes gennem membranen. Hvis der kun registreres et par PMV&#39;er under et regelmæssigt mikroskop ved hjælp af 10X-objektivet, eller hvis størrelsen af ​​PMV&...

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

Aldersrelaterede sygdomme er forbundet med flere defekter i cytoplasmaets komponenter. Her præsenterer vi en protokol til fremstilling af plasmamembranvesikler fra knoglemarvs mesenkymale stamceller. Denne teknik kan potentielt anvendes som et middel til cytoplasmudskiftningsterapi til at forbedre eller endog omvendt aldersrelaterede fænotyper.

Fremstilling af plasmamembresvesikler fra knoglemarv Mesenkym stamceller til potentiel cytoplasm erstatningsterapi

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

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