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.

Etik uttalande: Rutiner som involverar djurförsök har godkänts av Institutional Animal Care och användning kommittén vid Stanford University School of Medicine. OBS: Djur är under bedövning med 3% förångare isofluran och 3% leverans av O2 gas. Användning av veterinär salva på ögonen kan bidra till att förhindra torrhet under narkos. 1. Cellberedning <ol><li> Väx humana endotelceller och pericyte cellkulturer i 100 mm plattor med 10 ml av den lämpliga media. Ersätt 10 ml fär...

<ol>
	<li>Risau, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+angiogenesis.">Mechanisms of angiogenesis.</a> Nature. 386 (6626), 671-674 (1997).</li><li>Ribatti, D., Nico, B., Crivellato, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+pericytes+in+angiogenesis.">The role of pericytes in angiogenesis.</a> Int J Dev Biol. 55 (3), 261-268 (2011).</li><li>Koh, W., Stratman, A. N., Sacharidou, A., Davis, G. 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Matrisen gel plug-analys har visat sig vara en praktisk och kraftfull metod för att utvärdera genreglering i angiogenes, angiogena och antiangiogena föreningar in vivo, och att komplettera in vitro-tester. Här beskriver vi i detalj en ny matris gel plug-analys av humant angiogenes som undersöker samspelet mellan endotelceller och pericyter under kärlbildningen. Det finns några nya och viktiga steg i detta protokoll. Celler i låg passage (passage 1-4) är att föredr...

Angiogenes är den process genom vilken nya blodkärl bildas från ett befintligt vaskulära nätverket 1 och är i fokus för pågående forskning inom många områden som sträcker sig från normal utveckling till sjukdomen. Denna dynamiska process involverar proliferation och migration av endotelceller (ECS) och rekrytering av pericyter att konstruera en vaskulär rör som är riktad mot den plats som behöver syre och näringstillförsel 2. För att studera denna process kräver en lika dynamisk analys, framför allt en som kan sammanfatta den tredimensionella karaktären av röret bildning. In vitro 3D matrisanalyser har utvecklats för att tillgodose detta behov och har fungerat bra för att tillåta forskare att definiera diskreta steg i tid och rum där angiogenes sker 3, 4, 5, 6. Men dessa in vitro 3D matrismodeller begränsad till att studera icke-perfusion fartyg end saknar därför kritiska komponenter som är relevanta för angiogenesprocessen (t.ex. cirkulerande tillväxt och hämmande faktorer, onaturlig spänning / krafter över kärlbädden) och misslyckas med att simulera den komplexa miljö som finns i levande vävnad. Att ta itu med denna begränsning har flera in vivo angiogenesanalyser utvecklats 7, inklusive matrisgel kontakten analys som kommer att vara i fokus för vår rapport 8, 9. Matrisen gel plug-analysen är en väletablerad in vivo angiogenesanalys som tilltalar forskare eftersom det ger en robust plattform för att testa roller olika celler och ämnen i angiogenes. Matrisgel är ett kommersiellt tillgängligt basalmembran-lösning som utsöndras av Engelbreth-Holm-Swarm (EHS) mus sarkom-cellinje som stelnar till en gel-liknande material vid 37 ° C. Matrisen gel kan blandas med celler och / eller substanser, såsom tillväxtfaktorer och Injected subkutant i musen. Värd EC invaderar kontakten under 14 dagar, bildar en vaskulär nätverk, och blir perfusion med värdens blod. Hittills har matris gel plug-analyser fokuserat på studier av endotelceller beteende under angiogenes, dock, såvitt vi vet inga ansträngningar har ännu gjorts för att avgöra om denna analys kan användas för att co-kultur endotelceller och pericyter att studera hur dessa två celltyper samverkar under angiogenes. Specifikt är värdefull för att studera sjukdomar där blodkärl förlust är patologisk, inklusive mikrovaskulära ischemi och perifer kärlsjukdom 10, 11, 12 förstå sambandet mellan EC och pericyter. Här beskriver vi ett protokoll som introducerar pericyter humant ursprung till matrisen gelblandningen tillsammans med mänsklig EC och fibroblasttillväxtfaktor (bFGF). Denna blandning kan sedan injiceras subkutant in ryggen på SCID-möss för att möjliggöra bildandet av fullt fungerande, pericyte belagda, hybrid fartyg. Vår protokoll beskriver hur man förbereder matris gel pluggar innehållande humana EC antingen med eller utan mänskliga pericyter, placering i SCID-möss och hur man kan analysera histologiska sektioner för kritiska angiogenes ändpunkter.

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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.

Representant H &amp; E och immunofluorescerande färgning av matris gel plug sektioner visas i Figur 2. Avsnitten från EG bara pluggar visa vissa fartyg som oftast inte perfuserade med blod (Figur 2 uppe till vänster, svarta pilar) medan pluggar som innehåller både EC och pericyter visar flera perfunderad fartyg med större diameter och fullständig pericyte täckning, vilket framgår av positiv SMA färgning i direkt anslutning till CD31-positiva EC...

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

Vi presenterar ett protokoll för att studera mänskliga endotel-pericyte interaktioner i musen med en variation av matris gel kontakten angiogenes analys.

In vivo-studie av Human Endothelial-Pericyte interaktion med hjälp av Matrix Gel Plug-analys i mus

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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