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.

Ethikerklärung: Verfahren unter Verwendung tierischer Probanden wurden von der Institutional Animal Care und Use Committee an der Stanford University School of Medicine zugelassen. HINWEIS: Die Tiere sind unter anesthetization mit 3% Verdampfer Isofluran und 3% Zufuhr von O 2 -Gas. Die Verwendung von Tierarzt Salbe auf die Augen kann helfen, Trockenheit während der Narkose zu verhindern. 1. Zellpräparation <ol><li> Wachsen humanen Endothel- und Perizyten Zellkulturen in 100 mm-Platten mit...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+three+dimensional+collagen+matrix+models+of+endothelial+lumen+formation+during+vasculogenesis+and+angiogenesis.">In vitro three dimensional collagen matrix models of endothelial lumen formation during vasculogenesis and angiogenesis.</a> Methods Enzymol. 443, 83-101 (2008).</li><li>Liu, Y., Senger, 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=Matrix-specific+activation+of+Src+and+Rho+initiates+capillary+morphogenesis+of+endothelial+cells.">Matrix-specific activation of Src and Rho initiates capillary morphogenesis of endothelial cells.</a> FASEB J. 18 (3), 457-468 (2004).</li><li>Grant, D. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+modulation+of+endothelial+cells+by+transforming+growth+factor-beta+depends+upon+the+composition+and+organization+of+the+extracellular+matrix.">Phenotypic modulation of endothelial cells by transforming growth factor-beta depends upon the composition and organization of the extracellular matrix.</a> J Cell Biol. 106 (4), 1375-1384 (1988).</li><li>Brooks, P. C., Montgomery, A. M., Cheresh, D. 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Die Matrix Gelpfropfen Assay hat eine bequeme und leistungsfähige Methode zur Bewertung der Genregulation in Angiogenese, angiogene und antiangiogene Verbindungen in vivo erwiesen, und in vitro - Tests zu ergänzen. Hier beschreiben wir im Detail eine neuartige Matrix Gelpfropfen Test der menschlichen Angiogenese, die die Interaktion zwischen Endothelzellen und Perizyten während Gefäßbildung untersucht. Es gibt einige neuartige und wichtige Schritte in diesem Protokoll....

Die Angiogenese ist der Prozess , durch den neue Blutgefäße aus einem bereits bestehenden Gefäßnetz gebildet werden 1 und steht im Mittelpunkt der laufenden Forschung in vielen Bereichen von der normalen Entwicklung von Krankheiten reichen. Dieser dynamische Prozess beinhaltet die Proliferation und Migration von endothelialen Zellen (ECs) und die Rekrutierung von Perizyten eines Gefäßrohr zu konstruieren , die in Richtung auf die Seite gerichtet ist , die zwei Sauerstoff- und Nährstoffzufuhr benötigt. Zur Untersuchung dieser Prozess einen ebenso dynamischen Test erfordert, vor allem eine, die die dreidimensionale Natur der Schlauchbildung rekapitulieren kann. In - vitro - 3D - Matrix - Assays wurden entwickelt , um diesen Bedarf zu adressieren und gearbeitet haben und Forscher zu ermöglichen , die einzelnen Schritte in Raum und Zeit zu definieren , in denen die Angiogenese Platz 3 nimmt, 4, 5, 6. Allerdings sind diese in vitro 3D - Matrix - Modelle beschränkt auf das Studium nicht-durchbluteten Gefäßen eined fehlt daher kritische Komponenten relevant für die Prozess der Angiogenese (zB Wachstum zirkulierenden und hemmenden Faktoren, unnatürliche Spannung / Kräfte über das Gefäßbett) und nicht die komplexe Umwelt in lebendem Gewebe zu simulieren. Um dieser Einschränkung, mehrere in - vivo - Angiogenese - Assays wurden 7 entwickelt, einschließlich der Matrix Gelpfropfen Assay, der im Mittelpunkt unseres Berichts wird 8, 9. Die Matrix Gelpfropfen Assay ist ein etabliertes in vivo - Angiogenese - Test, der den Forschern anspricht , wie es eine robuste Plattform bietet die Rollen von verschiedenen Zellen und Substanzen in die Angiogenese zu testen. Matrix-Gel ist ein kommerziell erhältlicher Basalmembran Lösung, die von dem Engelbreth-Holm-Swarm (EHS) Maus-Sarkom-Zelllinie sekretiert wird, die bei 37 ° C in ein gelartiges Material erstarrt. Die Matrix-Gel kann mit Zellen und / oder Substanzen, wie Wachstumsfaktoren, gemischt werden und injesubkutan in die Maus cted. Der Host-ECs wird den Stecker über 14 Tage überfallen, ein Gefäßnetz bilden und mit dem Blut des Wirts durchblutet sind. Bislang Matrix Gelpfropfen Assays ausschließlich auf die Untersuchung der endothelialen Zellverhalten während der Angiogenese konzentriert sich jedoch auf bestem Wissen ist keine Anstrengung noch aus, um festzustellen, ob dieser Assay zur Kokultur Endothelzellen und Perizyten verwendet werden können, zu untersuchen, wie sich diese beiden Zelltypen während der Angiogenese zusammenwirken. Insbesondere ist wertvoll zwischen ECs und Perizyten die Beziehung Verständnis für Krankheiten zu studieren , wo Blutgefäßverlust pathologisch, einschließlich mikrovaskulären Ischämie und periphere Gefäßerkrankungen 10, 11, 12. Hier beschreiben wir ein Protokoll, das Perizyten Menschen stamm an die Matrix Gelmischung zusammen mit menschlichen ECs und Fibroblastenwachstumsfaktor (bFGF) einführt. Diese Mischung kann dann i subkutan injiziert werdenn die dorsum von SCID-Mäusen die Bildung von voll funktionsfähig, pericyte beschichtet, Hybridgefäße zu ermöglichen. Unser Protokoll beschreibt, wie Matrix Gelpfropfen, die menschliches ECs vorzubereiten entweder mit oder ohne menschliches pericytes, Platzierung in SCID-Mäuse und wie die histologischen Schnitte für kritische Angiogenese-Endpunkte zu analysieren.

<table border="0" cellpadding="0" cellspacing="0" width="806">
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	<tbody>
		<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.

Repräsentative H &amp; E und Immunfluoreszenzanfärbung von Matrix Gelpfropfen Abschnitte sind in Abbildung 2 Abschnitte von EG nur Stecker einige Schiffe angezeigt werden gezeigt, die meist nicht durchblutet (Abbildung 2 oben links, schwarze Pfeile) , während Stecker , die sowohl ECs und Perizyten Anzeige mehrere perfundierter Schiffe mit größerem Durchmesser und vollständige pericyte Abdeckung, wie durch positive SMA Färbung unmittelbar angrenzen...

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

Wir stellen ein Protokoll menschlichen endothelial-pericyte Wechselwirkungen in Maus unter Verwendung einer Variation der Matrix Gelpfropfen Angiogenese-Assay zu untersuchen.

In - vivo - Studie der menschlichen Endothelial-pericyte Interaktion Mit dem Matrix - Gel - Plug - Assay in der Maus

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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