Este trabalho foi apoiado pela concessão do National Institutes of Health (HL115578 para A. Makino).

A pesquisa em ratos foi aprovado pelo Comitê Institucional de Animal Care and Use (IACUC) da Universidade do Arizona e foi realizada de acordo com o Instituto Nacional de Saúde (NIH) orientações. NOTA: Seções 1, 2 e 3 devem ser realizadas um dia antes do experimento, como a instalação. 1. Soluções Preparando <ol><li> tampão de CD31 (50 ml) <ol><li> Adicionar 50 ml de 1 x tampão fosfato salino (PBS) a um tubo de 50 ml. Pesar 0,05 g de Albumina de Soro Bovino (BSA) e 0,037 g de ácido etileno...

<ol>
	<li>Michiels, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+functions.">Endothelial cell functions.</a> J Cell Physiol. 196, 430-443 (2003).</li><li>Sumpio, B. E., Riley, J. T., Dardik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+in+focus:+endothelial+cell.">Cells in focus: endothelial cell.</a> Int J Biochem Cell Biol. 34, 1508-1512 (2002).</li><li>Cines, D. B., 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=Endothelial+cells+in+physiology+and+in+the+pathophysiology+of+vascular+disorders.">Endothelial cells in physiology and in the pathophysiology of vascular disorders.</a> Blood. 91, 3527-3561 (1998).</li><li>Deanfield, J. E., Halcox, J. P., Rabelink, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+function+and+dysfunction:+testing+and+clinical+relevance.">Endothelial function and dysfunction: testing and clinical relevance.</a> Circulation. 115, 1285-1295 (2007).</li><li>Jin, Y., Liu, Y., Antonyak, M., Peng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+vascular+endothelial+cells+from+murine+heart+and+lung.">Isolation and characterization of vascular endothelial cells from murine heart and lung.</a> Methods Mol Biol. 843, 147-154 (2012).</li><li>Makino, A., Platoshyn, O., Suarez, J., Yuan, J. X., Dillmann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Downregulation+of+connexin40+is+associated+with+coronary+endothelial+cell+dysfunction+in+streptozotocin-induced+diabetic+mice.">Downregulation of connexin40 is associated with coronary endothelial cell dysfunction in streptozotocin-induced diabetic mice.</a> Am J Physiol Cell Physiol. 295, C221-C230 (2008).</li><li>Marelli-Berg, F. M., Peek, E., Lidington, E. A., Stauss, H. J., Lechler, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+murine+tissue.">Isolation of endothelial cells from murine tissue.</a> J Immunol Methods. 244, 205-215 (2000).</li><li>van Beijnum, J. R., Rousch, M., Castermans, K., vander Linden, E., Griffioen, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+endothelial+cells+from+fresh+tissues.">Isolation of endothelial cells from fresh tissues.</a> Nat Protoc. 3, 1085-1091 (2008).</li><li>Nolan, D. 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=Molecular+signatures+of+tissue-specific+microvascular+endothelial+cell+heterogeneity+in+organ+maintenance+and+regeneration.">Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.</a> Dev Cell. 26, 204-219 (2013).</li><li>Sobczak, M., Dargatz, J., Chrzanowska-Wodnicka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+pulmonary+endothelial+cells+from+neonatal+mice.">Isolation and culture of pulmonary endothelial cells from neonatal mice.</a> J Vis Exp. , (2010).</li><li>Makino, A., Dai, A., Han, Y., Youssef, K. D., Wang, W., Donthamsetty, R., Scott, B. T., Wang, H., Dillmann, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=O-GlcNAcase+overexpression+reverses+coronary+endothelial+cell+dysfunction+in+type+1+diabetic+mice.">O-GlcNAcase overexpression reverses coronary endothelial cell dysfunction in type 1 diabetic mice.</a> Am J Physiol Cell Physiol. 309, C593-C599 (2015).</li><li>Sasaki, K., Donthamsetty, R., Heldak, M., Cho, Y., Scott, B. T., Makino, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VDAC:+old+protein+with+new+roles+in+diabetes.">VDAC: old protein with new roles in diabetes.</a> Am J Physiol Cell Physiol. 303, C1055-C1060 (2012).</li></ol>

As células endoteliais mais utilizados na pesquisa são células humanas endoteliais da veia umbilical (HUVECs) por causa de sua conveniência e facilidade de cultivo. No entanto, uma grande quantidade de pesquisa requer a disponibilidade de um modelo fisiológico atingido pelo isolamento de células endoteliais específicas de outros órgãos 10. As células endoteliais formam uma população muito reduzida das células no tecido do coração; seu isolamento e cultura pode revelar-se muito difícil. <p ...

modelos de ratinho de várias doenças cardiovasculares e desordens metabólicas suportar mudanças fisiológicas e moleculares que são semelhantes aos encontrados em pacientes. Além disso, a alteração genética de ratinhos é uma ferramenta poderosa que nos permite investigar o papel patogénico de genes específicos no desenvolvimento e progressão de doenças. Hoje em dia, o gene-knock-in específico de células-tipo ou ratinhos -out podem ser facilmente gerados em muitos laboratórios e a medição de ARNm e os níveis de proteína em tipos específicos de células é o primeiro passo para determinar se os ratos foram verdadeiramente geneticamente modificado. O endotélio é uma camada única fina de células endoteliais (ECs) que reveste a parede vascular interior. As células endoteliais desempenham um papel crítico na regulação da tensão vascular, da permeabilidade vascular, e nova formação vascular 1,2. A disfunção endotelial é uma característica de muitas condições patológicas e alterações na função endotelial pode conduzir a vários carrosdoenças cardiovasculares 3,4. É, assim, importante para estudar a função das células endoteliais em condições fisiológicas e fisiopatológicas. Existem várias maneiras de isolar ECs 5-8 e o método de isolamento tem que ser optimizado dependendo de qual tecidos e espécies serão utilizados. Isto é porque CEs a partir de diferentes tecidos apresentam um elevado nível de heterogeneidade no que diz respeito aos seus marcadores de superfície e expressão da proteína 9. isolamento de células endoteliais pode muitas vezes ser um desafio e requer algum grau de treinamento e prática. Uma vez alcançada, o método de isolamento proposto neste protocolo revela-se estável e eficiente. O objetivo geral deste método é a obtenção de rato ECs coronariana (MCECs) de alta qualidade e quantidade. Embora a quantidade de células recolhidas a partir de um coração é menos do que as células recolhidas a partir de outros tecidos, utilizando outras técnicas, esta técnica ainda proporciona apostaqualidade ter. A pureza das células endoteliais na população resultante é maior do que 90%. Assim, essa técnica seria o ideal para aplicações que dependem de células endoteliais puramente isolados, como imagens de células.

<table border="0" cellpadding="0" cellspacing="0" width="876">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:239px;">BSA</td>
			<td style="width:251px;">Sigma</td>
			<td style="width:108px;">A3311-10G</td>
			<td style="width:280px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Fisher</td>
			<td>BP120-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>Fisher</td>
			<td>SH3026801</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepes</td>
			<td>Sigma-Aldrich</td>
			<td>H4034-500G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Bicarbonate&#160;</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-(+)-Glucose&#160;</td>
			<td>Sigma</td>
			<td>G7021-1KG</td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">Sheep anti-rat IgG beads&#160;</td>
			<td>Life Technologies</td>
			<td>F-410L</td>
			<td style="width:280px;">DynaBeads pan mouse IgG , Thermo Fisher Scientific, cat# 11035</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat anti-mouse PECAM Antibody&#160;</td>
			<td>BD Pharmigen</td>
			<td>550274</td>
			<td>BD Biosciences</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IFCS&#160;</td>
			<td>Fisher</td>
			<td>SH30072.04</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M199</td>
			<td>Fisher</td>
			<td>MT10060-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dispase (Neutral Protease)</td>
			<td>Worthington Biochemical Corp./Fisher</td>
			<td>LS02109</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase Type II&#160;</td>
			<td>Worthington Biochemical Corp./Fisher</td>
			<td>LS004176</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol&#160;</td>
			<td>Fisher</td>
			<td>BP381-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Igepal</td>
			<td>Sigma</td>
			<td>13021-50ml</td>
			<td>I3021-50 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphatase inhibitor cocktail</td>
			<td>Sigma</td>
			<td>P0044-1ml</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protease inhibitor cocktail</td>
			<td>Sigma</td>
			<td>P8340-1 ml</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin&#160;</td>
			<td>Sigma</td>
			<td>H3149-100KU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS&#160;</td>
			<td>Fisher/Mediatech</td>
			<td>MT35010CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-Valine</td>
			<td>Sigma</td>
			<td>V1255-5G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EGS</td>
			<td>BD</td>
			<td>356006</td>
			<td>Corning</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin</td>
			<td>Fisher/Mediatech</td>
			<td>MT-30-002-Cl</td>
			<td>MT-30-002-CI</td>
		</tr>
	</tbody>
</table>

isolation of mouse coronary endothelial cells

As células endoteliais estão alinhados na parede interna dos vasos sanguíneos e desempenham um papel importante na regulação do tónus vascular, da permeabilidade vascular, e nova formação vascular. disfunção das células endoteliais está implicada no desenvolvimento e progressão de muitas doenças cardiovasculares, incluindo doença cardíaca isquémica. Para examinar a função e caracterização de células endoteliais coronárias, o isolamento de células é o primeiro passo e exige elevado grau de pureza e a quantidade para conduzir experiências subsequentes. Este protocolo descreve um método eficiente para isolar ratinho adulto células endoteliais coronárias. O coração do rato é dissecado e picada em pedaços pequenos. Após a digestão do coração utilizando colagenase e dispase II, as células são lavadas e incubadas com esferas magnéticas que são conjugados com o anticorpo anti-CD31. As contas com células endoteliais são lavadas várias vezes e está pronto para usar em várias aplicações, incluindo imagens e experime biológica molecularNTS. Isolamento eficiente rende aproximadamente 10 4 células por um coração com mais de 90% de pureza.

O isolamento de células endoteliais coronárias de um coração de rato normalmente produz cerca de 10 4 células com mais de 90% de pureza com uma forma de paralelepípedos. Para uma população pura de células endoteliais, o cuidado deve ser tomado ao longo do protocolo para garantir um ambiente estéril livre de qualquer contaminação. O aparecimento de células alongadas ou células alargada dentro da população indica contaminação com outros tipos de células, as c?...

Watch this Scientific Journal Video about Isolation of Mouse Coronary Endothelial Cells at JoVE.com

Isolation of Mouse Coronary Endothelial Cells

This protocol is prepared to share our method of isolating mouse coronary endothelial cells for the purpose of imaging or to conduct molecular biological experiments.

Isolamento de células de camundongo Coronária endoteliais

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new ...

The overall goal of this experiment is to obtain large numbers of highly purified high quality mouse coronary endothelial cells. This method can help answer key questions in the field of cardiovascular research. The main advantage of this technique is that it facilitates the acquisition of a high yield of good quality endothelial cells.Demonstrating this procedure will be Shizhen Luo, a research specialist, and Dr.Ayako Makino, our principal investigator. Up to one week before the procedure, add one milliliter of CD31 buffer to one 1.5 milliliter microcentrifuge tube per sample. Next, mix sheep antirad IGG beads with gentle pipetting, and add the appropriate volume of beads to each tube.After all of the beads have been added, shake the tubes vigorously 30 times and place them on a magnetic plate for one minute of shaking. Then, with the tubes still attached to the plate, discard the buffer and add one milliliter of fresh CD31 buffer to each tube. Add two microliters of rad anti-mouse CD31 antibody and place the tubes on a rotator at four degrees Celsius overnight.The next day, transfer the tubes under a laminar flow hood and place them back onto the magnetic plate with shaking for one minute. Then replace the supernatant with one milliliter of fresh CD31 buffer and shake the tubes vigorously by hand 30 times. After the third wash, return the tubes to the rotator at four degrees Celsius.On the day of the procedure, begin by injecting the experimental animal intraperitoneally with 0.1 milliliters of heparin to prevent blood coagulation. After ten minutes, anesthetize the mouse with an injection of 0.01 milliliters pentobarbital. Then, confirm a lack of response to toe pinch.After removing the heart and lung tissue together, place them into a beaker of Krebs buffer and rinse the tissues with gentle shaking. Then transfer the tissues into a six centimeter dish containing fresh Krebs, and use scissors and foreceps to remove the lung lobes. Now insert a 20 gauge catheter into the aorta and flush the coronary circulatory system with HBSS supplemented with heparin.When all of the blood has been removed, make a small cut in the ventricle, rinse the heart with gentle shaking, and place the cardiac tissue into a sample tube on ice. After digesting the tissue to release the coronary endothelial cells, pellet and wash the cells two times with five milliliters of washing buffer. Then place a tube of the previously prepared beads onto the magnetic plate and shake the tube three times to evenly distribute the beads.After the second wash, remove as much supernatant as possible without disrupting the pellet, and associate the cells with vigorous flicking. Next, resuspend the cells in one milliliter of washing buffer with gentle mixing and discard the CD31 buffer from the beads. Combine the cells with the beads, and flick the tube 30 times.Then place the cell and bead solution onto the rotator at four degrees Celsius for 30 minutes. While the cells are incubating, coat the surface of one plating chamber well per sample with gelatin solution, and place the chambers at 37 degrees Celsius for 30 minutes. When the gelatin has solidified, remove any excess gel solution and dry the chamber well surface.Then transfer the bead conjugated cells to the magnetic plate under the hood for two minutes of shaking, and discard the washing buffer. Add one milliliter of fresh washing buffer to the cells and remove the tube from the plate for 30 vigorous shakes, followed by 30 flicks. Return the tube to the plate for another minute of shaking.After the last agitation, return the tube to the magnetic plate for a final minute of shaking, and replace the washing buffer with one milliliter of 37 degree Celsius endothelial cell medium, supplemented with 20 percent iron fortified calf serum. Shake and flick the tubes 30 final times. Then use a one milliliter pipetter set to 500 microliters to agitate the conjugated cells ten times, and transfer 500 microliter aliquots of coronary endothelial cells to each plating chamber well for overnight incubation at 37 degrees Celsius.The next day, wash the cells in endothelial cell medium, supplemented with ten percent FBS. The purity of the coronary endothelial cells isolated by this method can be confirmed by immunohistochemical staining against the endothelial cell surface markers, BS lectin, and AC LDL, with over 90 percent of the cells demonstrating a double positive expression of both markers in a typical mouse coronary endothelial cell culture. Following this procedure, as a method, various blood, immunoreception, and fluorescent imaging can be performed to answer additional questions, such as what protein can be modified during different disease states.After its development, this technique provides the tools necessary for researchers to expand their research into the field of coronary endothelial cells. After watching this video, you should have a good understanding of how to isolate mouse coronary endothelial cells of high quantity and quality. There are several key points during this procedure.The most important thing is to maintain a sterile environment throughout the isolation. Second is to flush the blood completely and as soon as possible to avoid blood coagulation in the capillary network. Third is to measure the pH of all the buffer, especially CD31 buffer.

isolation-of-mouse-coronary-endothelial-cells

Isolamento de células de camundongo Coronária endoteliais

Abstract