Name: in vivo study of human endothelial-pericyte interaction using the matrix gel plug assay in mouse
Uploaded: 2016-12-19T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse at JoVE.com

Dr. K. Yuan was supported by an American Heart Association Scientist Development Grant (15SDG25710448) and the Pulmonary Hypertension Association Proof of Concept Award (SPO121940). Dr. V. de Jesus Perez was supported by a career development award from the Robert Wood Johnson Foundation, an NIH K08 HL105884-01 award, a Pulmonary Hypertension Association Award, a Biomedical Research Award from the American Lung Association and a Translational Research and Applied Medicine award from Stanford University.

Ethiek Verklaring: Procedures waarbij proefdieren zijn goedgekeurd door de Institutional Animal Care en gebruik Comite aan de Stanford University School of Medicine. LET OP: De dieren zijn onder verdoving met 3% verdamper isofluraan en 3% levering van O 2 gas. Het gebruik van vet zalf op de ogen kan helpen tot droog te voorkomen dat tijdens het onder narcose. 1. Celbereiding <ol><li> Groeien humane endotheliale en pericyte celkweken in 100 mm platen met 10 ml van de geschikte media. Vervang ...

<ol>
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De matrix gel plug bepaling blijkt een gemakkelijke en krachtige methode om genregulatie in angiogenese, angiogene en anti-angiogene verbindingen in vivo te evalueren en in vitro testen kunnen vullen. Hier beschrijven we in detail een nieuwe matrix gel plug assay van humane angiogenese die de interactie tussen endotheliale cellen en pericyten onderzoekt bij bloedvatvorming. Er zijn een paar nieuwe en kritische stappen in dit protocol. Cellen in lage passage (passage 1-4) de...

Angiogenese is het proces waardoor nieuwe bloedvaten worden gevormd uit een reeds bestaande vasculaire netwerk 1 en is het onderwerp van onderzoek in vele gebieden, variërend van normale ontwikkeling ziekten. Dit dynamische proces omvat de proliferatie en migratie van endotheelcellen (EC) en de aanwerving van pericytes een vasculaire buis die is gericht op de site die zuurstof en voedingsstoffen levering 2 moet construeren. Studeren vereist deze werkwijze een even dynamische test, vooral degene die de driedimensionale aard van vaatvorming kan herhalen. In vitro 3D matrix assays ontwikkeld om deze behoefte en goed gefunctioneerd om onderzoekers de discrete stappen in ruimte en tijd waarin angiogenese plaatsvindt 3, 4, 5, 6 definiëren. Echter, deze in vitro 3D matrixmodellen beperkt tot het bestuderen van niet-geperfuseerde vaten ind daarom geen kritische componenten die relevant zijn voor het proces van angiogenese (bijv circulerende groei en remmende factoren, onnatuurlijke spanningen / krachten over het vasculaire bed) en niet aan de complexe omgeving aanwezig in levend weefsel te simuleren. Om deze beperking te pakken zijn verscheidene in vivo angiogenese assays ontwikkeld 7, waaronder de matrixgel plug assay die de focus van ons rapport 8, 9 zijn. De matrix gel plug test is een gevestigde in vivo angiogenese assay dat een beroep op de onderzoekers omdat het een robuust platform om de rol van de verschillende cellen en stoffen bij angiogenese te testen. Matrixgel is een commercieel verkrijgbare basaalmembraan oplossing die wordt afgescheiden door de Engelbreth-Holm-Swarm (EHS) muizen sarcoma cellijn die stolt tot een gel-achtig materiaal bij 37 ° C. De gel matrix kan worden gemengd met cellen en / of stoffen, zoals groeifactoren en injesubcutaan cted in de muis. De gastheer EC zal de plug binnenvallen meer dan 14 dagen, vormen een vasculair netwerk, en raken perfusie met bloed van de gastheer. Tot op heden zijn matrixgel plug assays uitsluitend gericht op de studie van endotheelcellen gedrag tijdens angiogenese echter om ons beste weten geen inspanningen nog uitgevoerd om te bepalen of deze assay kan worden gebruikt voor co-kweek endotheelcellen en pericyten te bestuderen hoe deze twee celtypen interactie tijdens angiogenese. Specifiek, inzicht in de relatie tussen EC en pericyten is waardevol voor het bestuderen van ziekten waarbij bloedvat verlies pathologisch, waaronder microvasculaire ischemie en perifere vaatziekte 10, 11, 12. We beschrijven een protocol dat humaan afgeleide pericytes introduceert de matrixgel mengsel samen met humane EC en fibroblast groeifactor (bFGF). Dit mengsel kan vervolgens subcutaan in het dorsale gedeelte van SCID muizen om de vorming van volledig functioneel, pericyte gecoat, hybride schepen toe te staan. Ons protocol beschrijft hoe matrix gel pluggen die humane EC met of zonder menselijke pericytes, plaatsing in SCID muizen en hoe de histologische secties voor kritische angiogenese eindpunten te analyseren voor te bereiden.

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		<tr height="21">
			<td height="21" style="height:21px;width:405px;">PBS (Phosphate buffered saline)</td>
			<td style="width:115px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">0.25% Trypsin/0.53mM EDTA</td>
			<td style="width:115px;">Corning</td>
			<td style="width:119px;">25-053-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Endothelial Cell Media (ECM) kit</td>
			<td style="width:115px;">Sciencell</td>
			<td style="width:119px;">1001</td>
			<td>includes media, EC growth supplement, and penicillin/streptomycin, each supplied at the appropriate volume for easy mixing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Pericyte Media (PM) kit</td>
			<td style="width:115px;">Sciencell</td>
			<td style="width:119px;">1201</td>
			<td>includes media, Pericyte growth supplement, and penicillin/streptomycin, each supplied at the appropriate volume for easy mixing</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:405px;">primary human pulmonary microvascular endothelial cells</td>
			<td style="width:115px;">Pulmonary Hypertension Breakthrough Initiative</td>
			<td style="width:119px;"></td>
			<td>this cell type is also available commercially. Cells used at passage 1-4</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:405px;">primary human pulmonary pericytes</td>
			<td style="width:115px;">Pulmonary Hypertension Breakthrough Initiative</td>
			<td style="width:119px;"></td>
			<td>this cell type is not available commercially, but brain paricytes are.&#160; Cells used at passage 1-4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">bFGF (basic Fibroblast Growth Factor)</td>
			<td style="width:115px;">Peprotech</td>
			<td style="width:119px;">100-18B</td>
			<td>stock solution is 50ug/ml in 0.1% BSA in PBS, aliquots at 50 uL and stored at -20 degrees</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Matrigel Basement Membrane Matrix</td>
			<td style="width:115px;">BD</td>
			<td style="width:119px;">356237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">28G 1cc Insluin Syringe</td>
			<td style="width:115px;">BD</td>
			<td style="width:119px;">329410</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">SCID (Severe Combined Immune Deficiency) mice</td>
			<td style="width:115px;">The Jackson Laboratory</td>
			<td>5557</td>
			<td>NOD.SCID IL2R gamma knockout strain is the best strain; 4-6 weeks of age</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">1.5mL microcentrifuge tubes, sterile</td>
			<td style="width:115px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">15 mL screw top tubes, sterile</td>
			<td style="width:115px;">BD Biosciences</td>
			<td style="width:119px;">352096</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">PAP pen</td>
			<td style="width:115px;">Life Technologies</td>
			<td style="width:119px;">8899</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">hemocytometer</td>
			<td style="width:115px;">ThermoFisher Scientific</td>
			<td style="width:119px;">02-671-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Nair hair removal cream</td>
			<td style="width:115px;">Walmart</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">anti-human CD31 primary antibody</td>
			<td style="width:115px;">LifeSpan Biosciences</td>
			<td>LS-B4737</td>
			<td>working solution is 1:50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-human Smooth Muscle actin CY3 primary fluorescent antibody</td>
			<td style="width:115px;">Sigma</td>
			<td>C6198</td>
			<td>working solution is 1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">goat anti-rabbit secondary antibody; 488/green</td>
			<td style="width:115px;">ThermoFisher Scientific</td>
			<td style="width:119px;">A-11008</td>
			<td>working solution is 1:250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Prolong Gold DAPI solution</td>
			<td style="width:115px;">Cell Signaling</td>
			<td style="width:119px;">8961S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">microscope slides</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">48300-047</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">no. 1.5 cover slips</td>
			<td style="width:115px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12-544-D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">citrate buffer 10x</td>
			<td style="width:115px;">Millipore</td>
			<td style="width:119px;">21545</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">extra fine surgical scissors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td style="width:119px;">14084-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:405px;">Formalin (paraformaldehyde)</td>
			<td style="width:115px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">245-685</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">Tissue cassettes</td>
			<td style="width:115px;">Simport</td>
			<td>M492-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:405px;">goat serum</td>
			<td style="width:115px;">Dako</td>
			<td style="width:119px;">X0907</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo study of human endothelial-pericyte interaction using the matrix gel plug assay in mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

Vertegenwoordiger H &amp; E en immunofluorescentie kleuring van matrix gel plug secties worden weergegeven in figuur 2. De artikelen van EG uitsluitend stekkers weer enkele schepen die meestal niet worden geperfuseerd met bloed (Figuur 2 linksboven, zwarte pijlen), terwijl pluggen die zowel EC en pericytes tonen verschillende geperfundeerde schepen met grotere diameters en complete pericyte dekking, zoals blijkt uit de positieve SMA kleuring direct grenz...

Watch this Scientific Journal Video about In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse at JoVE.com

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse (Video) | JoVE

We presenteren een protocol voor humane endotheliale pericyte-interacties in muizen te bestuderen onder toepassing van een variatie van de matrix gel plug angiogenese assay.

In vivo studie van humane endotheliale-pericyte Interaction Met behulp van de Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

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In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

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