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 arbejde blev støttet af tilskud DK078732 fra NIH (til PTF).

1. Adenovirale Transduktion og dyrkning af rotteøer <ol><li> Fremstilling af en 6-brønds ikke-vævskulturplade overtrukne plade ved tilsætning af 2 ml medium (RPMI 1640-medier indeholdende 8 mM glucose, 10% føtalt bovint serum, 50 enheder / ml penicillin og 50 ug / ml streptomycin) til det krævede antal brønde. For eksempel kan et typisk eksperiment kræver tre brønde, én hver i en ikke-virus-kontrol, en viruskontrol (f.eks GFP-udtrykkende adenovirus) og den eksperimentelle gruppe. </li><li> Opvarme pladen 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>

Oprettelse veje, der kan moduleres til at stimulere replikation og forbedre funktionen af ​​beta-cellerne er relevante for begge større former for diabetes. Fordi funktionelle beta-cellemasse er afhængig af eksistensen og funktionen af ​​insulinproducerende celler, vurdere disse faktorer samtidig har sine fordele. Denne protokol beskrives en strømlinet protokol for at identificere, hvorvidt overekspression eller undertrykkelse af et protein fører til ændringer i funktionel beta cellemasse in vitro,...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> RPMI 1640 medium </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-brønds plade </td><td> BD-Falcon </td><td> 35-1146 </td><td> Ikke-TC behandlede </td></tr><tr><td> [Methyl-3H]-thymidin </td><td> Perkin Elmer </td><td> NET027Z001MC </td><td> 1 mCi / ml </td></tr><tr><td> Mikro-centrifugerør </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> MgSO4 </td><td> Acros </td><td> 41348 </td><td></td></tr><tr><td> CaCl2 </td><td> Acros </td><td> 34961 </td><td></td></tr><tr><td> HEPES </td><td> Sigma </td><td> H0887 </td><td> 1 M opløsning </td></tr><tr><td> 35% BSA </td><td> Sigma </td><td> A7979 </td><td></td></tr><tr><td> NaHCO3 </td><td> Acros </td><td> 42427 </td><td></td></tr><tr><td> D-glucose </td><td> Sigma </td><td> G8769 </td><td></td></tr><tr><td> TCA </td><td> Fisher Scientific </td><td> SA9410-1 </td><td> 10% vægt / volumen </td></tr><tr><td> NaOH </td><td> Acros </td><td> 12426 </td><td></td></tr><tr><td> Scintillationstælling rør </td><td> Sarstedt </td><td> 58,536 </td><td> 7 ml, PP </td&gt; </tr><tr><td> Scintillationstælling rør hætte </td><td> Sarstedt </td><td> 65,816 </td><td></td></tr><tr><td> Econo-Safe tælle 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> Udstyr </td></tr><tr><td> Centrifuger </td><td> Eppendorf </td><td> 5415R </td><td></td></tr><tr><td> Scintillationstælling rør tandstang </td><td> Sarstedt </td><td> 93.1431.001 </td><td></td></tr><tr><td> Væskescintillationstæller </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

Glucosehomeostase styres primært af endokrine hormoner insulin og glucagon, secerneres fra pancreas-beta-og alfa-celler. Funktionel beta cellemasse bestemmes ved den anatomiske beta cellemassen såvel som evnen af ​​beta-cellerne til at reagere på en næringsstof. Et tab af funktionel beta-cellemasse er centralt for både store former for diabetes 1-3. De faldende funktionelle beta-cellemasse resultater fra en autoimmun angreb i type 1 diabetes, i type 2-diabetes denne decrement udvikler sig fra både en manglende evne til beta-celler til at udskille insulin hensigtsmæssigt og ødelæggelse af beta-celler fra en gruppe af mekanismer. Således bestræbelser på at genoprette funktionelle beta-cellemasse er altafgørende for den bedre behandling af og mulige behandlinger for diabetes. Bestræbelser i gang for at identificere molekylære reaktionsveje, der kan udnyttes til at stimulere replikation og forbedre funktionen af ​​beta-celler.Ideelt ville terapeutiske mål at forbedre både beta-celle vækst og funktion. Måske mere vigtigt er dog at identificere, om en strategi, der stimulerer beta cellevækst kommer på bekostning af svække beta-celle funktion (såsom med nogle onkogener) og vice versa. Ved systematisk at undertrykke eller overudtrykker ekspression af målgener i isolerede rotte-øvævsceller, kan man identificere potentielle terapeutiske mål for forøgelse funktionel beta cellemasse 4-6. Adenovirale vektorer kan anvendes til effektivt at overudtrykke eller knockdown proteiner i isolerede rotte-øvævsceller 4,7-15. Her præsenteres en fremgangsmåde til at manipulere genekspression anvender adenovirale transduktion og vurdere islet replikation og beta celle funktion i isolerede rotte-øvævsceller (figur 1). Denne fremgangsmåde er blevet anvendt tidligere til at identificere nye mål, som modulerer beta cellereplikation eller funktion 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 protokol tillader en at identificere faktorer, der modulerer funktionelle beta-cellemasse til at finde potentielle terapeutiske mål for behandling af diabetes. Den protokol består af en strømlinet metode til at vurdere islet replikation og beta celle funktion i isolerede rotte-øvævsceller efter manipulering af genekspression med adenovirus.

Vurdering Replication og betacellefunktionen i adenoviralt-transducerede Isolerede gnavere holme

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

Research

JoVE Journal

Medicine

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

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 ...

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 ...

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 ...

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 ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Evaluering Teratogene Ændringer i en Zebrafisk Model af Føtalt Alkohol Eksponering

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

Farvning protokoller for Menneskerettigheder bugspytkirteløer

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

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

Lepr<sup&gt; Db</sup&gt; Mus Model af type 2 diabetes: Pancreas Islet Isolering og live-cell 2-Photon Imaging af intakte 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, ...

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

A High-content In Vitro Pancreatic Islet ÃƒÅ½Ã‚Â²-cell Replication Discovery Platform

En High-indhold<em&gt; In vitro</em&gt; Pancreas Islet β-celle 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 ...

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

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

Kvantitativ 3D<em&gt; I Silico</em&gt; Modeling (q3DISM) af cerebral amyloid-beta Fagocytose i gnavermodeller af Alzheimers sygdom

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

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

Nerve-besparende Mid-urethral obstruktion (NEMO) i kvindelige lille Gnavere

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