Este trabajo fue apoyado por subvenciones del NIH DK078732 (para PTF).

1. Transducción adenoviral y el cultivo de islotes de rata <ol><li> Preparar un 6-así no tejido recubierto con placa de cultivo mediante la adición de 2 ml de medio (RPMI 1640 que contienen 8 mM de glucosa, 10% de suero fetal bovino, 50 unidades / ml de penicilina, y 50 ug / ml de estreptomicina) para el número requerido de pozos. Por ejemplo, un experimento típico puede requerir tres pozos, uno cada uno para un control sin virus, un control de virus (por ejemplo, GFP-expresión de adenovirus), y el grupo experimental. </li><li> Calentar la placa a 37 ° C colocándolo en una incubadora de cultivo de tejido por lo menos durante 30 min. </li><li> Inmediatamente después de aislamiento rata islote 18,19, 100-200 islotes lugar en pocillos individuales de la placa de cultivo de 6-bien no tejido revestido. Sesenta islotes son necesarios para la secreción de insulina y ensayos de incorporación de timidina. Los islotes restantes pueden ser utilizados para el aislamiento del ARN para estudios de expresión génica o el aislamiento de proteínas para inmunotransferencia. </li></ol> [Nota: A partir de este punto en adelante, por favor, siga los protocolos institucionales para el manejo, uso y desecho de materiales de riesgo biológico.] <ol start="4"><li> Girar suavemente la placa para llevar a los islotes al centro del pozo. </li><li> Pipetear el adenovirus directamente sobre los islotes en el centro del plato. Utilizar 100-500 multiplicidades de infección (MOI, la proporción de las células diana a virales unidades formadoras de placas). </li><li> Que el resto islotes durante 5 min. </li><li> Colocar la placa en el tejido incubadora de cultivo (37 ° C, 5% de CO 2). </li><li> Después de 24 h, agitar suavemente la placa para llevar a los islotes a los centros de los pozos y transferir los islotes utilizando una micropipeta P200 a un nuevo pozo que contiene medio fresco. Si los islotes se une a la placa, que puede ser desplazado suavemente con la punta de la pipeta. </li></ol> [Nota: Para verificar la efi transducción adecuadaCY, el uso de un virus de control que expresa GFP es beneficioso, como islotes entonces se pueden obtener imágenes a través de microscopía confocal para verificar la penetración del adenovirus en el núcleo de islotes.] <ol start="9"><li> Cultura de los islotes de un adicional de 24 a 72 h, en función del tiempo deseado de la experiencia de los estudios piloto de optimización. Por ejemplo, la inducción de una respuesta proliferativa puede requerir tiempos que van desde 24 hasta 72 horas o desmontables del gen de interés puede requerir 48 o 72 horas. Traslado de los islotes a medio fresco cada día. </li><li> Para el final de las 24 horas del experimento, la cultura de los islotes en los medios de comunicación que contienen 1 Ci [metil-3H] -thymidine/ml los medios de comunicación (generalmente de 1 l de timidina / ml de los medios de comunicación). </li></ol> [Nota: A partir de este punto en adelante, por favor, siga los protocolos institucionales para el manejo, uso y disposición de materiales radiactivos.] 2. La insulina secreción de ensayo <ol><li> Preparar elsecreción tampón de ensayo (SAB) solución madre 10 veces (1,14 M NaCl, 47 mM KCl, 12 mM de KH 2 PO 4, 11,6 mM de MgSO 4) y CaCl 2 solución madre 100X (0,25 M CaCl 2). Estas soluciones madre se pueden preparar antes de tiempo y se almacenan a temperatura ambiente. </li><li> Recién preparar 50 ml de la SAB de trabajo (5 ml de 10X SAB, 1 ml de HEPES 1 M, 0,5 ml de CaCl 100X 2, 0,28 ml de 35% de BSA, 0,11 g de NaHCO 3, y agua estéril para 50 ml) en un de 50 ml cónica del tubo y se calienta a 37 ° C, colocando en un baño de agua a 37 ° C. </li><li> Pipeta 10 ml de la SAB de trabajo en un tubo cónico de 15 ml y añadir 66,8 l de 2,5 M de D-glucosa para preparar la glucosa alta (16,7 mM) SAB. </li><li> Añadir 44,8 l de 2,5 M de D-glucosa a los restantes 40 ml de la SAB de trabajo para preparar la baja de glucosa (2,8 mM) SAB. </li><li> Etiqueta de tres de 1,7 ml tubos de microcentrífuga para cada pocillo de la placa de 6 pocillos y añadir 1 ml de buffer fosfato salino (PBS). </li></ol&gt; [Nota: A medida que los islotes son radiactivos, por favor, siga los protocolos institucionales para el manejo, uso y disposición de materiales radiactivos.] <ol start="6"><li> Coloque 20 islotes en cada tubo de microcentrífuga. Haga todo lo posible para añadir islotes de tamaño comparable a cada tubo de microcentrífuga. Por ejemplo, cada tubo puede contener 5 de pequeño, mediano 10, y 5 de gran tamaño islotes (véase Figura 1). </li></ol> [Nota: Los islotes se pueden visualizar utilizando un estereoscopio de disección o un microscopio estándar.] <ol start="7"><li> Después de que los islotes se han asentado en la parte inferior del tubo por gravedad (~ 2 min), aspirar el PBS con una micropipeta y desechar. </li></ol> [Nota: Como alternativa a la sedimentación por gravedad, los tubos pueden ser centrifugadas a 300 xg durante 1 min.] <ol start="8"><li> Para la pre-incubación, añadir 400 l de la glucosa bajaSAB glucosa, colocar los tubos (con sus tapas abiertas) en el tejido incubadora de cultivo (37 ° C, 5% de CO 2), y pre-incubar durante 60 min. Aspirar el SAB pre-incubación de baja la glucosa y los descartes. </li><li> Para la secreción de insulina basal, añadir 400 l de la glucosa baja SAB, colocar los tubos (con sus tapas abiertas) en el tejido incubadora de cultivo (37 ° C, 5% de CO 2), y se incuba durante 60 min. Recoger la baja de glucosa en SAB y ahorrar para el radioinmunoensayo de insulina. </li><li> Para la secreción de insulina estimulada, añadir 400 l de la glucosa alta SAB, colocar los tubos (con sus tapas abiertas) en el tejido incubadora de cultivo (37 ° C, 5% de CO 2), y se incuba durante 60 min. Recoger la alta glucosa en la SAB y ahorrar para el radioinmunoensayo de insulina. </li></ol> 3. Ensayo de incorporación de timidina <ol><li> Añadir 1 ml de PBS, después de los islotes se han asentado en la parte inferior del tubo por gravedad, aspirar el PBS con una micropipeta, desechar, y repetir esteEntra una vez. </li><li> Añadir 500 l de ácido hielo frío tricloroacético (TCA, 10% w / v) y se incuba en hielo durante 30 min. </li><li> Centrifugar los tubos a 16 000 g durante 3 min a 4 ° C. </li><li> Aspirar el TCA, añadir 80 l de 0,3 N NaOH, y se incuba durante 30 min a temperatura ambiente. Durante este tiempo, vigorosamente vórtice las muestras para s 5-10 cada 10 min. </li><li> Agregar 4 ml de cóctel de conteo de Econo-seguro para 7 tubos de centelleo ml de líquido de recuento. </li><li> Añadir 50 l de la muestra al tubo de centelleo contando, tapar el tubo, agitar brevemente, y cuentan en un contador de centelleo líquido. </li><li> Medir la concentración de proteína utilizando el ácido bicinconínico (BCA) de ensayo y 10 ul de muestra de acuerdo con el protocolo del fabricante. </li></ol> 4. Análisis de Datos <ol><li> Lleve a cabo el radioinmunoensayo de insulina siguiendo el protocolo del fabricante. </li><li> Normalizar la secreción de insulina y los datos de la incorporación de timidina con el concentrado de proteínaentration. </li></ol> 5. Los resultados representativos Un ejemplo del experimento para evaluar la replicación de islotes y la función de las células beta en los islotes de rata se muestra en la Figura 2. Este ejemplo muestra que la sobreexpresión adenoviral de la hipotética &quot;Gene # 6&quot; con firmeza estimula la replicación del islote, sin alterar la función celular beta. En el panel superior, los resultados del ensayo demuestran que la incorporación de timidina aumentar la expresión de &quot;Gene # 6&quot; aumenta la síntesis de ADN, medida por la incorporación de timidina. Dado que la mayoría de las células en el islote de rata son células beta, es probable que este aumento de la incorporación de timidina indica un aumento en la replicación de las células beta. Sin embargo, los experimentos de confirmación se debe realizar para establecer firmemente este. En el panel inferior, los resultados de la prueba de la secreción de insulina demuestran que la sobreexpresión de &quot;Gene # 6&quot; no alteró una de las funciones celulares primarias beta, es decir, insulin la secreción de la glucosa en baja y alta. La calidad del aislamiento de islotes y la salud de los islotes después del tratamiento con adenovirus se indica por el aumento de veces en la secreción de insulina en concentraciones de glucosa de baja y alta. Si el aumento de la expresión de &quot;Gene # 6&quot; alteración de la función de las células beta, esto es probable que se refleja como una disminución en la insulina secretada en altas concentraciones, estimuladores de glucosa (16,7 mM). Una curva dosis-respuesta para variar las concentraciones de glucosa también podría llevarse a cabo. <img src="/files/ftp_upload/4080/4080fig1.jpg" alt="Figura 1"/> Figura 1. Visión general de protocolo para evaluar la replicación del islote y la función de células beta en los islotes aislados de ratas después de mediada por adenovirus alteraciones en la expresión génica. Recién aislados islotes de rata están expuestos a adenovirus durante 24 horas y después se cultivaron hasta 96 h. La incorporación de timidina se evalúa en la final de 24 h, seguido por la medición de la secreción de insulina ala glucosa baja y alta. <img src="/files/ftp_upload/4080/4080fig2.jpg" alt="Figura 2"/> Figura 2. Los resultados de un experimento utilizando un adenovirus control y un adenovirus que sobreexpresan un gen hipotético etiquetado como &quot;Gene # 6&quot;. El panel superior muestra la incorporación de timidina y el panel inferior de la secreción de insulina.

<ol>
	<li>Ferrannini, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Cell+function+in+subjects+spanning+the+range+from+normal+glucose+tolerance+to+overt+diabetes:+a+new+analysis.">beta-Cell function in subjects spanning the range from normal glucose tolerance to overt diabetes: a new analysis.</a> J. Clin. Endocrinol. Metab. 90, 493-500 (2005).</li><li>Weyer, C., Bogardus, C., Mott, D. M., Pratley, R. 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+natural+history+of+insulin+secretory+dysfunction+and+insulin+resistance+in+the+pathogenesis+of+type+2+diabetes+mellitus.">The natural history of insulin secretory dysfunction and insulin resistance in the pathogenesis of type 2 diabetes mellitus.</a> J. Clin. Invest. 104, 787-794 (1999).</li><li>Keenan, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+insulin+production+and+pancreatic+ss-cell+turnover+after+50+years+of+diabetes:+Joslin+Medalist+Study.">Residual insulin production and pancreatic ss-cell turnover after 50 years of diabetes: Joslin Medalist Study.</a> Diabetes. 59, 2846-2853 (2010).</li><li>Bain, J. R., Schisler, J. C., Takeuchi, K., Newgard, C. B., Becker, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+adenovirus+vector+for+efficient+RNA+interference-mediated+suppression+of+target+genes+in+insulinoma+cells+and+pancreatic+islets+of+langerhans.">An adenovirus vector for efficient RNA interference-mediated suppression of target genes in insulinoma cells and pancreatic islets of langerhans.</a> Diabetes. 53, 2190-2194 (2004).</li><li>Fueger, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trefoil+factor+3+stimulates+human+and+rodent+pancreatic+islet+beta-cell+replication+with+retention+of+function.">Trefoil factor 3 stimulates human and rodent pancreatic islet beta-cell replication with retention of function.</a> Mol. Endocrinol. 22, 1251-1259 (2008).</li><li>Schisler, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+human+and+rat+islet+beta-cell+proliferation+with+retention+of+function+by+the+homeodomain+transcription+factor+Nkx6.1.">Stimulation of human and rat islet beta-cell proliferation with retention of function by the homeodomain transcription factor Nkx6.1.</a> Mol. Cell Biol. 28, 3465-3476 (2008).</li><li>Chan, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+uncoupling+protein+2+inhibits+glucose-stimulated+insulin+secretion+from+rat+islets.">Overexpression of uncoupling protein 2 inhibits glucose-stimulated insulin secretion from rat islets.</a> Diabetes. 48, 1482-1486 (1999).</li><li>Cozar-Castellano, I., Takane, K. K., Bottino, R., Balamurugan, A. N., Stewart, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+beta-cell+proliferation+and+retinoblastoma+protein+phosphorylation+in+rat+and+human+islets+using+adenovirus-mediated+transfer+of+cyclin-dependent+kinase-4+and+cyclin+D1.">Induction of beta-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of cyclin-dependent kinase-4 and cyclin D1.</a> Diabetes. 53, 149-159 (2004).</li><li>Icyuz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+infection+activates+akt1+and+induces+cell+proliferation+in+pancreatic+islets1.">Adenovirus infection activates akt1 and induces cell proliferation in pancreatic islets1.</a> Transplantation. 87, 821-824 (2009).</li><li>Kaneto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+hexosamine+pathway+leads+to+deterioration+of+pancreatic+beta-cell+function+through+the+induction+of+oxidative+stress.">Activation of the hexosamine pathway leads to deterioration of pancreatic beta-cell function through the induction of oxidative stress.</a> J. Biol. Chem. 276, 31099-31104 (2001).</li><li>Antinozzi, P. A., Berman, H. K., O'Doherty, R. M., Newgard, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+engineering+with+recombinant+adenoviruses.">Metabolic engineering with recombinant adenoviruses.</a> Annu. Rev. Nutr. 19, 511-544 (1999).</li><li>Newgard, C. B., Becker, T. C., Berman, H. K., O'Doherty, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+overexpressed+hexokinases+in+liver+and+islet+cells.">Regulation of overexpressed hexokinases in liver and islet cells.</a> Biochem. Soc. Trans. 25, 118-122 (1997).</li><li>Becker, T. C., BeltrandelRio, H., Noel, R. J., Johnson, J. H., Newgard, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+hexokinase+I+in+isolated+islets+of+Langerhans+via+recombinant+adenovirus.+Enhancement+of+glucose+metabolism+and+insulin+secretion+at+basal+but+not+stimulatory+glucose+levels.">Overexpression of hexokinase I in isolated islets of Langerhans via recombinant adenovirus. Enhancement of glucose metabolism and insulin secretion at basal but not stimulatory glucose levels.</a> J. Biol. Chem. 269, 21234-21238 (1994).</li><li>Csete, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral-mediated+gene+transfer+to+pancreatic+islets+does+not+alter+islet+function.">Adenoviral-mediated gene transfer to pancreatic islets does not alter islet function.</a> Transplant Proc. 26, 756-757 (1994).</li><li>Csete, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+to+pancreatic+islets+mediated+by+adenoviral+vectors.">Efficient gene transfer to pancreatic islets mediated by adenoviral vectors.</a> Transplantation. 59, 263-268 (1995).</li><li>Meng, Z. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+liver+X+receptors+inhibits+pancreatic+islet+beta+cell+proliferation+through+cell+cycle+arrest.">Activation of liver X receptors inhibits pancreatic islet beta cell proliferation through cell cycle arrest.</a> Diabetologia. 52, 125-135 (2009).</li><li>Ronnebaum, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyruvate+cycling+pathway+involving+cytosolic+NADP-dependent+isocitrate+dehydrogenase+regulates+glucose-stimulated+insulin+secretion.">A pyruvate cycling pathway involving cytosolic NADP-dependent isocitrate dehydrogenase regulates glucose-stimulated insulin secretion.</a> J. Biol. Chem. , (2006).</li><li>Milburn, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+beta-cells+in+obesity.+Evidence+for+induction+of+functional,+morphologic,+and+metabolic+abnormalities+by+increased+long+chain+fatty+acids.">Pancreatic beta-cells in obesity. Evidence for induction of functional, morphologic, and metabolic abnormalities by increased long chain fatty acids.</a> J. Biol. Chem. 270, 1295-1299 (1995).</li><li>Szot, G., Koudria, P., Bluestone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+Pancreatic+Islet+Isolation.">Murine Pancreatic Islet Isolation.</a> J. Vis. Exp. (7), e255 (2007).</li></ol>

No hay conflictos de interés declarado.

El establecimiento de las vías que pueden ser moduladas para estimular la replicación y mejorar la función de las células beta son relevantes para las dos formas principales de diabetes. Debido a que la masa de células beta funcionales depende de la existencia y función de las células secretoras de insulina, la evaluación de estos factores determinantes al mismo tiempo tiene sus ventajas. Este protocolo se describe un protocolo simplificado para determinar si la sobreexpresión o la supresión de una proteína c...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> RPMI 1640 medios </td><td> Gibco </td><td> 11879 </td><td></td></tr><tr><td> La penicilina / estreptomicina </td><td> Gibco </td><td> 15140 </td><td></td></tr><tr><td> 6-así placa </td><td> BD-Falcón </td><td> 35-1146 </td><td> No tratada TC </td></tr><tr><td> [Metil-3 H]-timidina </td><td> Perkin Elmer </td><td> NET027Z001MC </td><td> 1 mCi / ml </td></tr><tr><td> Centrífuga micro-tubos </td><td> Denville </td><td> C2170 </td><td> 1,7 ml </td></tr><tr><td> NaCl </td><td> Sigma </td><td> 59888 </td><td></td></tr><tr><td> KCl </td><td> Acros </td><td> 42409 </td><td></td></tr><tr><td> KH 2 &lt;/ Sub&gt; PO 4 </td><td> Acros </td><td> 20592 </td><td></td></tr><tr><td> MgSO 4 </td><td> Acros </td><td> 41348 </td><td></td></tr><tr><td> CaCl 2 </td><td> Acros </td><td> 34961 </td><td></td></tr><tr><td> HEPES </td><td> Sigma </td><td> H0887 </td><td> 1 M solución </td></tr><tr><td> 35% de BSA </td><td> Sigma </td><td> A7979 </td><td></td></tr><tr><td> NaHCO $ 3 </td><td> Acros </td><td> 42427 </td><td></td></tr><tr><td> d-glucosa </td><td> Sigma </td><td> G8769 </td><td></td></tr><tr><td> TCA </td><td> Fisher Scientific </td><td> SA9410-1 </td><td> 10% w / v </td></tr><tr><td> NaOH </td><td> Acros </td><td> 12426 </td><td></td></tr><tr><td> Centelleo contando tubo </td><td> Sarstedt </td><td> 58.536 </td><td> 7 ml, PP </td&gt; </tr><tr><td> Centelleo tubo de contar la tapa </td><td> Sarstedt </td><td> 65.816 </td><td></td></tr><tr><td> Econo-Safe cóctel de contar </td><td> RPI </td><td> 111175 </td><td></td></tr><tr><td> La insulina RIA </td><td> Siemens </td><td> TKIN2 </td><td></td></tr><tr><td> Kit de ensayo de BCA </td><td> Thermo Scientific </td><td> 23250 </td><td></td></tr><tr><td></td><td></td><td></td><td> Equipo </td></tr><tr><td> Centrifugar </td><td> Eppendorf </td><td> 5415R </td><td></td></tr><tr><td> Recuento de centelleo gradilla </td><td> Sarstedt </td><td> 93.1431.001 </td><td></td></tr><tr><td> Contador de centelleo líquido </td><td> Perkin Elmer </td><td> Tri-Carb 2910TR </td><td></td></tr></tbody></table>

assessing replication and beta cell function in adenovirally-transduced isolated rodent islets

Homeostasis de la glucosa se controla principalmente por la insulina y glucagon endocrino hormonas, secretada por la beta del páncreas y las células alfa, respectivamente. Masa funcional de las células beta está determinada por la masa de células beta anatómica, así como la capacidad de las células beta para responder a una carga de nutrientes. Una pérdida de masa funcional de las células beta es fundamental para ambas formas principales de diabetes 1-3. Considerando que los resultados la disminución de masa de células beta funcionales de un ataque autoinmune en la diabetes tipo 1, diabetes tipo 2, este decremento se desarrolla a partir tanto de la incapacidad de las células beta que segregan la insulina de manera adecuada y la destrucción de las células beta de un grupo de mecanismos. Por lo tanto, los esfuerzos para restaurar la masa funcional de las células beta son fundamentales para el tratamiento y la mejor de las curas potenciales para la diabetes. Se están realizando esfuerzos para identificar las vías moleculares que pueden ser aprovechados para estimular la replicación y mejorar la función de las células beta.Idealmente, los objetivos terapéuticos que mejoraría tanto el crecimiento de las células beta y su función. Quizás lo más importante es sin embargo para identificar si una estrategia que estimula el crecimiento de las células beta es a costa de perjudicar la función de las células beta (por ejemplo, con algunos oncogenes) y viceversa. En forma sistemática supresión o la sobreexpresión de la expresión de los genes diana en islotes aislados de rata, se puede identificar posibles dianas terapéuticas para el aumento de la masa funcional de las células beta 6.4. Los vectores adenovirales pueden ser empleados para las proteínas eficientemente sobreexpresan o desmontables en los islotes de rata aislados 4,7-15. A continuación, presentamos un método para manipular la expresión génica utilizando transducción adenoviral y evaluar la replicación del islote y la función de células beta en los islotes aislados de rata (Figura 1). Este método ha sido utilizado anteriormente para identificar nuevos objetivos que modulan la replicación de las células beta o de la función 5,6,8,9,16,17.

Watch this Scientific Journal Video about Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets at JoVE.com

Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Este protocolo permite a uno identificar los factores que modulan la masa funcional de las células beta para encontrar posibles dianas terapéuticas para el tratamiento de la diabetes. El protocolo consiste en un método simplificado para evaluar la replicación de islotes y la función de las células beta en los islotes de rata aislados siguientes manipulación de la expresión génica con adenovirus.

La evaluación de la replicación y la función celular beta en Adenovirally transducidas aislados islotes de roedores

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, ...

assessing-replication-beta-cell-function-adenovirally-transduced

Research

JoVE Journal

Medicine

13.9K vistas.  Indiana University School of Medicine. Este protocolo permite a uno identificar los factores que modulan la masa funcional de las células beta para encontrar posibles dianas terapéuticas para el tratamiento de la diabetes. El protocolo consiste en un método simplificado para evaluar la replicación de islotes y la función de las células beta en los islotes de rata aislados siguientes manipulación de la expresión génica con adenovirus.

Video: Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

Evaluación de la función cardiaca y Energética en los corazones de ratón aislado utilizando 31 P espectroscopía de RMN

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

Investigación

Medicina

Assessing Endothelial Vasodilator Function with the Endo-PAT 2000

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of the panoply of paracrine factors that it secretes, the endothelium regulates the contractile and proliferative state of the underlying vascular smooth muscle, as well as the interaction of the vessel wall with circulating blood elements. Because of its central role in mediating vessel tone and growth, its position as gateway to circulating immune cells, and its local regulation of hemostasis and coagulation, the the properly functioning endothelium is the key to cardiovascular health. Conversely, the earliest disorder in most vascular diseases is endothelial dysfunction.
In the arterial circulation, the healthy endothelium generally exerts a vasodilator influence on the vascular smooth muscle. There are a number of methods to assess endothelial vasodilator function. The Endo-PAT 2000 is a new device that is used to assess endothelial vasodilator function in a rapid and non-invasive fashion. Unlike the commonly used technique of duplex ultra-sonography to assess flow-mediated vasodilation, it is totally non-operator-dependent, and the equipment is an order of magnitude less expensive. The device records endothelium-mediated changes in the digital pulse waveform known as the PAT ( peripheral Arterial Tone) signal, measured with a pair of novel modified plethysmographic probes situated on the finger index of each hand. Endothelium-mediated changes in the PAT signal are elicited by creating a downstream hyperemic response. Hyperemia is induced by occluding blood flow through the brachial artery for 5 minutes using an inflatable cuff on one hand. The response to reactive hyperemia is calculated automatically by the system. A PAT ratio is created using the post and pre occlusion values. These values are normalized to measurements from the contra-lateral arm, which serves as control for non-endothelial dependent systemic effects. Most notably, this normalization controls for fluctuations in sympathetic nerve outflow that may induce changes in peripheral arterial tone that are superimposed on the hyperemic response.
In this video we demonstrate how to use the Endo-PAT 2000 to perform a clinically relevant assessment of endothelial vasodilator function.

A noninvasive procedure to assess endothelial function is demonstrated using the Endo-PAT 2000.

Valoración de la función endotelial vasodilatador con el 2000 Endo-PAT

The endothelium is a delicate monolayer of cells that lines all blood vessels, and which comprises the systemic and lymphatic capillaries. By virtue of ...

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

Myo-mecánico Análisis de músculo esquelético aislado

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

El aislamiento de islotes humanos de pacientes parcialmente Pancreatectomized

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Evaluación de los cambios teratogénicos en un modelo de pez cebra de la exposición fetal al alcohol

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Protocolos de tinción de islotes pancreáticos humanos

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Lepr<sup&gt; Db</sup&gt; Modelo de ratón de la Diabetes Tipo 2: El aislamiento de los islotes pancreáticos y células en Vivo 2-Photon imágenes de islotes intactos

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &amp;#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

Un alto contenido<em&gt; In Vitro</em&gt; Islotes pancreáticos de células β plataforma de descubrimiento de replicación

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer&#039;s Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

3D cuantitativa<em&gt; In silico</em&gt; Modelado (q3DISM) de beta-amiloide cerebral fagocitosis en roedores modelos de enfermedad de Alzheimer

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

Preservación de los nervios de mitad de obstrucción uretral (NEMO) en femenino pequeños roedores

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...