Name: assessing replication and beta cell function in adenovirally-transduced isolated rodent islets
Uploaded: 2012-06-25T13:00:00
Description: Watch this Scientific Journal Video about Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets at JoVE.com

Dette arbeidet ble støttet av stipend DK078732 fra NIH (til PTF).

1. Adenoviral Transduksjon og dyrkning av Rat Holmer <ol><li> Forbered en 6-brønnen ikke-vevskultur belagt plate ved å legge 2 ml media (RPMI 1640 medier som inneholder 8 mm glukose, 10% fosterets bovint serum, 50 enheter / ml penicillin, og 50 mikrogram / ml streptomycin) til det nødvendige antall brønner. For eksempel kan en typisk eksperiment krever tre brønner - én hver for et nei-virus kontroll, et virus kontroll (f.eks GFP-uttrykke adenovirus), og den eksperimentelle gruppen. </li><li> Varm opp platen til ...

<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>

Etablering veier som kan være modulert å stimulere replikering og forbedre funksjonen av beta-cellene er relevante for både store former for diabetes. Fordi funksjonell beta cellemasse er avhengig av eksistens og funksjon av insulin-sekresjon celler, vurdere disse determinantene har samtidig sine fordeler. Denne protokollen beskriver en strømlinjeformet protokoll for å identifisere hvorvidt overuttrykte eller undertrykkelse av et protein fører til endringer i funksjonell beta cellemasse in vitro, som dere...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> RPMI 1640 media </td><td> Gibco </td><td> 11879 </td><td></td></tr><tr><td> Penicillin / streptomycin </td><td> Gibco </td><td> 15140 </td><td></td></tr><tr><td> 6-brønnen plate </td><td> BD-Falcon </td><td> 35-1146 </td><td> Ikke-TC behandlet </td></tr><tr><td> [Metyl-3 H]-tymidin </td><td> Perkin Elmer </td><td> NET027Z001MC </td><td> 1 mCi / ml </td></tr><tr><td> Micro-sentrifugerør </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 løsning </td></tr><tr><td> 35% 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-glukose </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> Scintillation telle tube </td><td> Sarstedt </td><td> 58.536 </td><td> 7 ml, PP </td&gt; </tr><tr><td> Scintillation telle tube cap </td><td> Sarstedt </td><td> 65.816 </td><td></td></tr><tr><td> Econo-Safe telle cocktail </td><td> RPI </td><td> 111175 </td><td></td></tr><tr><td> Insulin RIA </td><td> Siemens </td><td> TKIN2 </td><td></td></tr><tr><td> BCA Assay Kit </td><td> Thermo Scientific </td><td> 23250 </td><td></td></tr><tr><td></td><td></td><td></td><td> Utstyr </td></tr><tr><td> Sentrifuger </td><td> Eppendorf </td><td> 5415R </td><td></td></tr><tr><td> Scintillation telle tube stativ </td><td> Sarstedt </td><td> 93.1431.001 </td><td></td></tr><tr><td> Flytende scintillation teller </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

Glukose homeostase er først og fremst styres av endokrine hormoner insulin og glukagon, skilles fra bukspyttkjertelen beta og alpha celler, henholdsvis. Funksjonell beta cellemasse bestemmes av den anatomiske beta cellemasse samt evne betacellene til å svare på et næringsstoff belastning. Et tap av funksjonell beta cellemasse er sentral i begge hovedformene av diabetes 1-3. Mens de fallende funksjonelle beta cellemasse resultater fra en autoimmun angrep i type 1 diabetes, i type 2 diabetes utvikler dette Decrement fra både en manglende evne til beta-cellene til å skille ut insulin riktig og ødeleggelse av betaceller fra en kadre av mekanismer. Dermed arbeidet med å restaurere funksjonell beta cellemasse er ytterst viktig for bedre behandling av og potensielle botemidler for diabetes. Det arbeides med å identifisere molekylære mekanismer som kan utnyttes til å stimulere replikering og forbedre funksjonen av beta-cellene.Ideelt sett ville terapeutiske mål å forbedre både beta cellevekst og funksjon. Kanskje mer viktig om er å identifisere om en strategi som stimulerer beta cellevekst kommer på bekostning av svekke betacellefunksjon (for eksempel med noen onkogener) og vice versa. Ved systematisk å undertrykke eller overekspresjon uttrykket av målgener i isolerte rotte holmer, kan man identifisere potensielle terapeutiske mål for å øke funksjonell beta cellemasse 4-6. Adenoviral vektorer kan brukes til å effektivt overuttrykker eller knockdown proteiner i isolerte rotte holmer 4,7-15. Her presenterer vi en metode for å manipulere genuttrykket utnytte adenoviral transduksjon og vurdere holme replikering og betacellefunksjon i isolerte rotte holmer (figur 1). Denne metoden har blitt brukt tidligere til å identifisere nye mål som modulerer beta cellen replikering eller funksjon 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

Denne protokollen gjør det mulig å identifisere faktorer som modulerer funksjonell beta cellemasse å finne potensielle terapeutiske mål for behandling av diabetes. Protokollen består av en strømlinjeformet metode for å vurdere holmen replikering og betacellefunksjon i isolerte rotte holmer etter manipulering av genuttrykk med adenoviruses.

Vurdere Replication og betacellefunksjon i Adenovirally-transduced Isolert gnager Holmer

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

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