Name: a murine pancreatic islet cell-based screening for diabetogenic environmental chemicals
Uploaded: 2018-06-25T12:30:00.000+00:00
Duration: 7 min 39 s
Description: Watch this Scientific Journal Video about A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals at JoVE.com

Diese Arbeit wurde durch ein Pilotprojekt Stipendium CREH Zentrum gesponsert durch NIEHS und National Natural Science Foundation of China (Nr. 31572626) unterstützt.

Alle Tierversuche wurden hingerichtet, unter Beachtung aller relevanten Richtlinien, Verordnungen und Aufsichtsbehörden. Das Protokoll wird gezeigt wurde unter der Leitung und Genehmigung der institutionellen Animal Care und Nutzung Committee (IACUC) von der Texas A & M Institut für Genomic Medizin durchgeführt.
1. Vorbereitung der Lösung
<ol><li>10 X Hank ausgeglichene Salzlösung, 1 X mit doppelt destilliertem H2O verdünnen und Lagerung bei 4 ° C.</li>
	<li>Bereiten Sie die Isolierungslösung indem HEPES (10 mM), MgCl2 (1 mM) und Glukose (5 mM). Einstellen Sie den pH-Wert 7,4 und halten Sie auf dem Eis.</li>
	<li>Bereiten Sie die Kollagenase-Lösung von Kollagenase Typ 4. Hinweis: Insbesondere enthält Kollagenase Typ 4 160 U pro mg Trockengewicht. 1 g Kollagenase Typ 4 wird in 5 mL doppelt destilliertem H2O mit einer Konzentration von 200 mg/mL verdünnt. 2 mL der verdünnten Kollagenase ist 30 mL Eis kalt isoliert Projektmappe hinzugefügt, und 6 mL Kollagenase-Lösung wird in ein 50 mL-Tube für jede Maus übertragen.</li>
	<li>Bereiten Sie die Waschlösung durch Zugabe von 1 mM CaCl2 zur Isolierungslösung.</li>
	<li>Bereiten Sie die Reinigung Lösung mit kalten Polysucrose/Natrium-Diatrizoate-Lösung (1,1119 g/mL, 5 mL).</li>
	<li>Bereiten Sie 500 mL Kulturmedium Inselchen durch Zugabe von L-Glutamin (20 mM), Penicillin (100 U/mL), Streptomycin (100 µg/mL) und fetalen bovine Serum (FBS) (10 %) in RPMI Medium vor.</li>
	<li>Vorbereiten der RLKV-DA-Stammlösung durch Auflösen des Pulvers in destilliertem Wasser bei 200 µM und Store bei-20 ° C. Hinweis: Die Färbelösung ROS wird durch Verdünnung RLKV-DA-Stammlösung auf die Endkonzentration von 5 µM in RPMI frisch zubereitet. Dieses Reagenz sollten vor Licht geschützt werden.</li>
</ol>2. chirurgische Vorbereitung
<ol><li>Intraperitoneale Injektion (i.p.) eine Maus mit avertin (2,5 % avertin, 0,4 mL/20 g). Nach die Maus völlig betäubt ist und nicht mehr reagiert zum hinteren Fuß drückt, einschläfern die Maus und die biologische Motorhaube.</li>
	<li>Platzieren Sie die euthanasierten Maus mit dem Bauch nach oben und sprühen Sie die Haut mit 70 % igem Ethanol für die Sterilisation.</li>
	<li>Öffnen der Bauchhöhle mit einem 1 cm U-Schnitt auf den Oberbauch aus- und Einfahren der Haut in Richtung rostral über der Brust, die Bauchhöhle verfügbar zu machen.</li>
</ol>3. die Bauchspeicheldrüse Perfusion und Entfernung
<ol><li>Wie in Abbildung 1dargestellt, finden Sie den Standort der Ampulle. Verwenden Sie ein paar gebogene Pinzetten, Klemmen Sie den Zwölffingerdarm nahe der Position des Sphincter Oddi. Verwenden Sie ein weiteres Paar gebogene Pinzetten, Klemmen Sie die Zwölffingerdarm Wand um den Weg der Galle in den Zwölffingerdarm zu blockieren. Hinweis: Der Ampulle wurde nicht in diesem Schritt eingefügt. Dieser Schritt soll die Galle Weg in den Zwölffingerdarm blockieren.</li>
	<li>Positionieren Sie die Maus mit dem Kopf proximalen und distalen in Bezug auf die Forscher die Füße.</li>
	<li>Finden Sie den hauptgallengang und injizieren Sie langsam 3 mL Kollagenase-Lösung mit einer 30 G Nadel und 5 mL Spritze. Sicherstellen Sie, dass der Gallengang und Bauchspeicheldrüse nach der Injektion aufgeblasen werden.</li>
	<li>Entfernen Sie die aufgeblähten Bauchspeicheldrüse mit den gebogenen Pinzette und feine Schere, um es von den absteigenden Dickdarm, Darm, Magen und Milz zu trennen. Die Bauchspeicheldrüse in einen 50-mL-Röhrchen mit 3 mL Kollagenase-Lösung und lassen Sie ihn auf Eis.</li>
</ol>4. Bauchspeicheldrüse Verdauung
<ol><li>Legen Sie je 50-mL-Tube in einem 37 ° C-Wasserbad. 15-20 min inkubieren (Zeit variiert je nach Sorte und Alter der Mäuse).</li>
	<li>Schwingen Sie das Rohr mit einem Wirbel.</li>
	<li>Pool 4-5 verdaute Bauchspeicheldrüse in ein großes sterile Waschbecken Sieb und eine Kugel verwenden, um die Bauchspeicheldrüse gründlich auseinander zu fallen. Verwenden Sie eine 23 G Nadel und 10 mL Spritze mit Waschlösung, um mit Nachdruck die verdauten Zellen außerhalb des Bildschirms pipette. Sammeln Sie die Waschlösung in einen 50-mL-Tube.</li>
	<li>Zentrifugieren Sie die Rohre mit 97 X g bei 4 ° C für 1 min und Dekantieren Sie des Überstandes.</li>
	<li>Rohre fügen Sie 25 mL Waschlösung hinzu und erneut aussetzen Sie des Gewebes durch sanfte aufschütteln. Spin-down der Gewebe bei 97 X g bei 4 ° C für 1 min und den überstand abgießen. Für 3 Mal wiederholen. Achten Sie darauf, dass Sie nicht das gebeizte Gewebe verdrängen.</li>
</ol>5. Reinigung der kleinen Inseln
<ol><li>Das gewaschene Gewebe Pellet 10 mL Reinigung Lösung hinzu und Aufschwemmen Sie sanft des Inhalts.</li>
	<li>Überlagern Sie sanft 5 mL Lösung zur Isolierung auf die Reinigung Lösung. Achten Sie darauf, um eine scharfe flüssigen Schnittstelle zwischen den beiden Lösungen zu halten.</li>
	<li>Zentrifugieren Sie das Rohr auf 560 x g bei 4 ° C für 15 min mit der langsamen Beschleunigung und keine Bremse. Legen Sie die Pipette in die Schnittstelle und sammeln Sie die Inselchen Schicht in neuen 50 mL Röhrchen.</li>
	<li>Waschen Sie die kleinen Inseln, wie unter Punkt 3.5 beschrieben.</li>
</ol>(6) Beschichtung und Behandlung von Inselzellen
<ol><li>Die kleinen Inseln in 10 mL Inselchen Kulturlösung Aufschwemmen, sanft gut mischen und 100 µL/Well mit einer Mehrkanal-Pipette auf eine 96-Well-Platte zu platzieren. Platzieren Sie ca. 5 Inseln pro 96 gut.</li>
	<li>Legen Sie die Platte in der Gewebekultur-Inkubator für 12 h.</li>
	<li>Zentrifugieren Sie (112 X g) 96-Well-Platte und entfernen Sie das Kulturmedium zu.</li>
	<li>Verdünnen Sie die Umweltchemikalien in das Kulturmedium Inselchen. 100 µL der einzelnen chemischen Verdünnungsmitteln in separaten Vertiefungen der Platte hinzufügen. Aufschwemmen der Inseln in jede Vertiefung und inkubieren Sie für 12 h bei 37 ° C (die Konzentration variiert für verschiedenen Umweltchemikalien).</li>
</ol>(7) ROS Färbung und Messung
<ol><li>Nachbehandlung der Inselchen mit 1 X PBS waschen (200 µL für jede 96-Well). Hinweis: Die Konzentration der Chemikalien ist wie folgt: AFB1: 3 µM, Atrazin: 100 µM, BaP: 10 µM, BPA: 100 µM, β-Östrogen, PCB126: 10 µM, Nonylphenol und BPA: 100 µM.</li>
	<li>Verwenden Sie N-Acetyl-Cystein (NAC) als Antioxidans ROS zu stillen. Die ROS-induzierten chemischen Verdünnungsmittel 5 mM der NAK hinzu und 12 h bei 37 ° c inkubieren</li>
	<li>Die 96-Well-Platte bei 112 X g zentrifugieren und mit 5 µM-Lösung von fluoreszierenden Sonde (H2-RLKV-DA aufgelöst in RPMI Medien) bei 37 ° C für 60 min inkubieren.</li>
	<li>Die behandelten Inselzellen 3 Mal mit PBS waschen und Messung die Fluoreszenz mit Dozent Mikrotestplatte an 488 nm. Hinweis: Die Anhäufung von DCF in Zellen kann gemessen werden, durch eine Erhöhung der Fluoreszenz bei 530 nm, wenn die Probe bei 488 angeregt wird nm.</li>
	<li>Führen Sie die Glukose stimuliert Insulin-Sekretion (GSIS) Assay9 auf ROS-induzierende Harnblase behandelt Inselchen und unbehandelten Inselchen, die insbesondere Auswirkungen auf die physiologische Funktion der Bauchspeicheldrüsen Inselchen zu messen.</li>
</ol>

<ol>
	<li>Maruthur, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growing+prevalence+of+type+2+diabetes:+increased+incidence+or+improved+survival?.">The growing prevalence of type 2 diabetes: increased incidence or improved survival?.</a> Current diabetes reports. 13 (6), 786-794 (2013).</li><li>Houstis, N., Rosen, E. D., Lander, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+have+a+causal+role+in+multiple+forms+of+insulin+resistance.">Reactive oxygen species have a causal role in multiple forms of insulin resistance.</a> Nature. 440 (7086), 944-948 (2006).</li><li>Ma, Z. A., Zhao, Z., Turk, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+and+beta-cell+failure+in+type+2+diabetes+mellitus.">Mitochondrial dysfunction and beta-cell failure in type 2 diabetes mellitus.</a> Exp Diabetes Res. , 703538 (2012).</li><li>Valavanidis, A., Vlahogianni, T., Dassenakis, M., Scoullos, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biomarkers+of+oxidative+stress+in+aquatic+organisms+in+relation+to+toxic+environmental+pollutants.">Molecular biomarkers of oxidative stress in aquatic organisms in relation to toxic environmental pollutants.</a> Ecotoxicology and environmental safety. 64 (2), 178-189 (2006).</li><li>Robertson, R. P., Harmon, J., Tran, P. O., Tanaka, Y., Takahashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+toxicity+in+&#946;-cells:+type+2+diabetes,+good+radicals+gone+bad,+and+the+glutathione+connection.">Glucose toxicity in &#946;-cells: type 2 diabetes, good radicals gone bad, and the glutathione connection.</a> Diabetes. 52 (3), 581-587 (2003).</li><li>Kaneto, H., 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=Oxidative+stress+induces+p21+expression+in+pancreatic+islet+cells:+possible+implication+in+beta-cell+dysfunction.">Oxidative stress induces p21 expression in pancreatic islet cells: possible implication in beta-cell dysfunction.</a> Diabetologia. 42 (9), 1093-1097 (1999).</li><li>Maechler, P., Jornot, L., Wollheim, 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=Hydrogen+peroxide+alters+mitochondrial+activation+and+insulin+secretion+in+pancreatic+beta+cells.">Hydrogen peroxide alters mitochondrial activation and insulin secretion in pancreatic beta cells.</a> Journal of Biological Chemistry. 274 (39), 27905-27913 (1999).</li><li>Gao, D., 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=The+effects+of+palmitate+on+hepatic+insulin+resistance+are+mediated+by+NADPH+Oxidase+3-derived+reactive+oxygen+species+through+JNK+and+p38MAPK+pathways.">The effects of palmitate on hepatic insulin resistance are mediated by NADPH Oxidase 3-derived reactive oxygen species through JNK and p38MAPK pathways.</a> Journal of Biological Chemistry. 285 (39), 29965-29973 (2010).</li><li>Efendi&#263;, S., 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=Pancreastatin+and+islet+hormone+release.">Pancreastatin and islet hormone release.</a> Proceedings of the National Academy of Sciences. 84 (20), 7257-7260 (1987).</li><li>Tian, Y., Ke, S., Denison, M. S., Rabson, A. B., Gallo, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ah+receptor+and+NF-&#954;B+interactions,+a+potential+mechanism+for+dioxin+toxicity.">Ah receptor and NF-&#954;B interactions, a potential mechanism for dioxin toxicity.</a> Journal of Biological Chemistry. 274 (1), 510-515 (1999).</li><li>Cui, H., 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=Pregnane+X+receptor+regulates+the+AhR/Cyp1A1+pathway+and+protects+liver+cells+from+benzo-[&#945;]-pyrene-induced+DNA+damage.">Pregnane X receptor regulates the AhR/Cyp1A1 pathway and protects liver cells from benzo-[&#945;]-pyrene-induced DNA damage.</a> Toxicology Letters. 275, 67-76 (2017).</li><li>Li, L. A., Wang, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCB126+induces+differential+changes+in+androgen,+cortisol,+and+aldosterone+biosynthesis+in+human+adrenocortical+H295R+cells.">PCB126 induces differential changes in androgen, cortisol, and aldosterone biosynthesis in human adrenocortical H295R cells.</a> Toxicological Sciences. 85 (1), 530-540 (2005).</li><li>Asahi, 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=Bisphenol+A+induces+endoplasmic+reticulum+stress-associated+apoptosis+in+mouse+non-parenchymal+hepatocytes.">Bisphenol A induces endoplasmic reticulum stress-associated apoptosis in mouse non-parenchymal hepatocytes.</a> Life sciences. 87 (13), 431-438 (2010).</li><li>Szot, G. L., Koudria, P., Bluestone, J. A. <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> JoVE (Journal of Visualized Experiments). (7), e255 (2007).</li><li>Kirstetter, P., Lagneau, F., Lucas, O., Krupa, Y., Marty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+endothelium+in+the+modulation+of+isoflurane-induced+vasodilatation+in+rat+thoracic+aorta.">Role of endothelium in the modulation of isoflurane-induced vasodilatation in rat thoracic aorta.</a> British journal of anaesthesia. 79 (1), 84-87 (1997).</li><li>Brown, E., Umino, Y., Loi, T., Solessio, E., Barlow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+can+cause+sustained+hyperglycemia+in+C57/BL6J+mice.">Anesthesia can cause sustained hyperglycemia in C57/BL6J mice.</a> Visual neuroscience. 22 (5), 615-618 (2005).</li><li>Vaupel, D., McCoun, D., Cone, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phencyclidine+analogs+and+precursors:+rotarod+and+lethal+dose+studies+in+the+mouse.">Phencyclidine analogs and precursors: rotarod and lethal dose studies in the mouse.</a> Journal of Pharmacology and Experimental Therapeutics. 230 (1), 20-27 (1984).</li><li>Neuman, J. C., Truchan, N. A., Joseph, J. W., Kimple, 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=A+method+for+mouse+pancreatic+islet+isolation+and+intracellular+cAMP+determination.">A method for mouse pancreatic islet isolation and intracellular cAMP determination.</a> Journal of visualized experiments: JoVE. (88), (2014).</li></ol>

Die Autoren erklären, dass sie keine finanziellen Interessenkonflikte.

Sammeln Beweise deutet darauf hin, dass die Exposition zu Umweltchemikalien spielt eine wichtige Rolle bei der Entwicklung des T2DM. XENOBIOTIKA-induzierte ROS wurde als ein möglicher ätiologischer Faktor zur Entwicklung des T2DM anerkannt. Menschen ein breites Spektrum von XENOBIOTIKA Chemikalien ausgesetzt sind, und es gibt ein großer Bedarf an neuartigen Forschungstechniken, effektiv die Bauchspeicheldrüse Giftstoffe zu identifizieren und zu untersuchen, den Mechanismus der Toxizität für die Zellen der Bauchspei...

Anstieg der Prävalenz von T2DM geworden in den letzten Jahren stellt eine ernsthafte Bedrohung für öffentliche Gesundheit1einer weltweiten Gesundheitskrise. Viele Faktoren gefunden worden zur Entwicklung des T2DM, darunter kausal verknüpft werden wiederkehrende Ergebnisse deuten darauf hin, dass eine konvergente Gemeinsamkeit dieser Faktoren ist die Induktion von oxidativem Stress führt zu der Generation von übermäßigen ROS2 , 3.
Ein breites Spektrum von Umweltchemikalien wie PCB, Dioxine und BaP hat sich herausgestellt, induzieren oxidativen Stress beeinträchtigen die Funktion der Bauchspeicheldrüse β-Zellen und zu Insulin-Resistenz und T2DM4führen kann. Obwohl der physiologische Ebene von ROS in zellulären Funktionen eine wichtige Rolle spielt, ROS, die die Kapazität des antioxidativen Systems übersteigt führt zu Schäden an Zellen/Gewebe und führt zu Krankheiten5. Pankreatische β-Zellen express ein niedriges Niveau der antioxidativen Enzym, und somit ist eine sensible Zielgruppe für den oxidativen Stress-vermittelten Schäden6,7. Chronische Exposition gegenüber hohen Konzentrationen von ROS wurde gezeigt, dass Stress-induzierte Pankreas Zelle Dysfunktion5 verursachen sowie Insulinresistenz in der Leber und Fettgewebe8.
Das übergeordnete Ziel dieses Projektes ist es, eine zellbasierte Assays Bildschirm Chemikalien für ihre diabetogene Potenziale basierend auf ihre Induktion von ROS in Zellen der Bauchspeicheldrüse entwickeln. Die Bauchspeicheldrüse fehlt metabolische Entgiftung und ist ein sensibler Ziel für Fremdstoff-induzierten Schäden6,7. Daher soll dieser Assay durch direkte Messung der ROS erzeugt in den Zellen der Bauchspeicheldrüsen, eine direkte Annäherung an die chemisch induzierte Schädigung der Bauchspeicheldrüse. Um diese Methode zu entwickeln, wir Maus Pankreas Inseln isoliert, kultiviert die isolierte Insel unter Zelle Kultur Bedingung mit Chemikalien und genutzt, die chemisch induzierte ROS-Generation als das Auslesen. Dieses Verfahren ist einfach und effektiv bei der Identifizierung von ROS-induzierende Chemikalien in der isolierten Insel; Es kann für die Untersuchung der Mechanismen der Toxizität weiterentwickelt werden, die speziell für die Bauchspeicheldrüse in Vitro.

<table><tbody><tr><td>10&amp;times;Hank&amp;rsquo;s balanced salt solution&amp;nbsp;</td><td>GIBCO</td><td>14185-052</td><td></td></tr><tr><td>Collagenase Type 4</td><td>Worthington Biochemical Corporation</td><td>CLS-4</td><td></td></tr><tr><td>polysucrose/sodium diatrizoate solution&amp;nbsp;</td><td>Sigma</td><td>10771</td><td></td></tr><tr><td>2&amp;rsquo;,7&amp;rsquo;-dichlorofluorescein (DCFH-DA)</td><td>Sigma</td><td>D6883-50MG</td><td></td></tr><tr><td>fluorescence microplate reader&amp;nbsp;</td><td>Biotek</td><td></td><td></td></tr><tr><td>L-glutamine</td><td>Sigma</td><td>G8540-25G</td><td></td></tr><tr><td>streptomycin</td><td>GIBCO</td><td>15140148</td><td></td></tr><tr><td>FBS</td><td>GIBCO</td><td>26140079</td><td></td></tr><tr><td>RPMI 1640</td><td>GIBCO</td><td>11875-085</td><td></td></tr><tr><td>avertin</td><td>Sigma</td><td>T48402-25G</td><td></td></tr><tr><td>Rat/Mouse Insulin ELISA Kit</td><td>Millipore</td><td>EZRMI-13K</td><td></td></tr><tr><td>Centrifuge</td><td>Sorval</td><td>Sorval RT7</td><td>for 96-well plate centrifuge</td></tr><tr><td>Microplate reader</td><td>Biotek</td><td>Epoch 2</td><td>for fluorescence reading</td></tr></tbody></table>

a murine pancreatic islet cell-based screening for diabetogenic environmental chemicals

Exposition gegenüber bestimmten Umweltchemikalien in Mensch und Tier wurde gefunden, um zelluläre Schäden der Bauchspeicheldrüse β-Zellen verursachen, die zu die Entwicklung von Diabetes Mellitus Typ 2 (T2DM) führt. Obwohl die Mechanismen für die chemisch induzierte β Zellschädigung unklar und wahrscheinlich zu komplex waren, ist eine immer wiederkehrende Erkenntnis, dass diese Chemikalien verursachen oxidativen Stress führt zu der Generation von übermäßigen reaktiven Sauerstoffspezies (ROS) die Schäden zu verursachen die β-Zelle. Ermittlung potenzieller diabetogene Umweltchemikalien, isoliert wir pankreatische Inselzellen von C57BL/6 Mäusen und kultivierten Inselzellen in 96-Well Zellplatten Kultur; dann wurden die Inselzellen mit Chemikalien dosiert und die ROS-Generation wurde von 2', 7'-dichlorofluoresceins (RLKV-DA) Fluoreszenzfarbstoff erkannt. Mit dieser Methode wir gefunden, dass Bisphenol A (BPA), Benzo [a] pyren (BaP) und polychlorierten Biphenylen (PCB), können hohe Konzentrationen von ROS, was darauf hindeutet, dass sie potenziell Schaden in Inselzellen induzieren können induzieren. Diese Methode sollte für das screening von diabetogene XENOBIOTIKA nützlich sein. Darüber hinaus können die kultivierten Inselzellen auch für in-vitro- Analyse der chemisch induzierten Toxizität in Zellen der Bauchspeicheldrüse angepasst werden.

Ein Schliffbild der gesunden isolierten Insel ist in Abbildung 2gezeigt, in dem kleinen Inseln eine Runde oder ovale Form mit relativ einheitliche Größe haben (obwohl Größe Einheitlichkeit von Stamm zu Stamm unterschiedlich sein kann). Wir als Nächstes untersucht pancreatic Islet-Funktionen in einer in-vitro- Assay durch das Inselchen zu isolieren und Stimulierung der Insulinsekretion in den Kultur-Inseln. Abbildung 3</stron...

Watch this Scientific Journal Video about A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals at JoVE.com

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Hier präsentieren wir ein Protokoll zur Maus pankreatische Inselzellen für das screening von ROS Induktionen durch die XENOBIOTIKA Ermittlung der potenziellen diabetogene XENOBIOTIKA Chemikalien zu isolieren.

Ein murinen Pancreatic Islet Cell-basierte Screening für diabetogene Umweltchemikalien

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

a-murine-pancreatic-islet-cell-based-screening-for-diabetogenic

Research

JoVE Journal

Immunology and Infection

7.2K Aufrufe.  Hunan Agriculture University. Hier präsentieren wir ein Protokoll zur Maus pankreatische Inselzellen für das screening von ROS Induktionen durch die XENOBIOTIKA Ermittlung der potenziellen diabetogene XENOBIOTIKA Chemikalien zu isolieren.

Serie: A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Murine Pancreatic Islet Isolation

Hi, I am Greg Zott. I'm with the UCSF Diabetes Center in the Bluestone Laboratory, and today I'm gonna show you the process for isolating mouse eyelets. The procedure involves the injection of a collagenase solution into the pancreas of a C3 H donor mouse, where we will then remove that pancreas, digest it away, purify the eyelets on a fial gradient, and then handpick them for ultimate transplant into the kidney capsule of a diabetic NOD mouse, Which will be our recipient.Before we start this procedure, we diluted our ATE solution in a modified Hanks buffer and we have adjusted the concentration specific for the strain of mouse that we're using the C3 H.We found digests better at a 0.8 mgs per mil collagenase solution, and we will use that to inject into the common bile duct to distend the pancreas.Performing. The procedure today Will be Pavo codre of the diabetes center. He will inject, open up the mouse, expose the the intestines, and then inject into the common bile duct.We will spray down the mouse with ethanol to keep the hair down, and then we will also verify that the mouse has been sacrificed properly with a toe pinch. Pavo will now open up the mouse and exposing the intestine and the pancreas with a midline incision. The diaphragm will also be cut to verify that the mouse will be sacrificed.Before we get started, I'd like to point out some of the important structures. The stomach is lying right here with the pancreas attached underneath into the duodenum and above. This is the liver where we'll find the gallbladder and the common bile duct, which you can see right here.So what Pavo will do next is he will isolate the small intestine, find where the common bile duct enters the small intestine. Once the intestine is exposed, we clamp on either side of the common bile duct with mosquito clamps. We find that this allows more collagenates to enter the pancreas while other groups try to direct the clamp on top of the common bile duct.We find that this is a better technique in allowing enzyme to enter the pancreas. This is a gentle procedure. You don't want to tear the bowel.You wanna allow the clamps to lie gently on either side of the common bile duct. Now we will find a gallbladder, and once the gallbladder's exposed, we will use that as a guide to injecting the collagenase into the common bile duct. Powell's using a 30 gauge needle with a five cc syringe containing three mills of collagenase solution.As he's entering the common bile duct, he's distending that duct with collagenase solution and then he travels down the common bile duct till he reaches near the intestine. And as he's going down, the enzyme solutions being pumped into the injected into the pancreas. Now that the pancreas is inflated with collagenase solution, Powell's gonna remove the hemostats and then remove the pancreas from the the mouse carefully tearing it away from the intestine.He removes it from the stomach. Okay, and then using the spleen as a handle, he lifts up the pancreas and then slowly snips it away in the common bile duct, and that's it. Pancreas is laid out and then the spleen is removed, and there he's removing the meso enteric tissue, and then the pancreas is placed in a two mill collagenase solution and a sterilized glass vial and it's placed back on Ice.The isolated pancreas that we put into the glass vials will now be placed into a 37 degree water bath for approximately 17 minutes. We placed them into a 50 mil conical rack so that they don't float up. Once during the bath, the timer starts and the digestion begins.Once the digestion time is completed, vials are removed, and then they are shaken and disrupted gently. And once the tissue starts falling apart, the vial is then placed on ice. A sterile screen is placed over a 400 mil beaker that contains washing buffer.The pancreas digest is then carefully poured over the screen into the wash buffer. For demonstration purposes, the hood has been turned off and the glass of the hood has been raised so we could see what we're doing. Once the pancreas digest is poured into the beaker, the vials are rinsed with the same washing buffer and the wash buffer placed over the screen.We have a syringe filled with washing buffer. We will use that to forcefully wash the tissue through the screen. The idea is to release any loose eyelets that are unattached to the matrix, but leaving the membranes and the undigested tissue on the screen.And when you're finished washing a tissue, this is what you usually See at the end of the procedure. The Pancreas tissue that's in the bottom of the tube will now be transferred to two 50 mil conical, and the tissue will be washed, the tissue's mixed evenly, and then distributed evenly so that we have a equal pellet volume in each tube. The beaker is now rinsed with more washing buffer.The sides are rinse so that you get all tissue that is entered the beaker again. Then it's equally distributed between each two, both 50 mil. Conical then are topped off with wash buffer.The tissue is inverted several times and then placed into a centrifuge. This is a quick spin. The centrifuge is allowed to reach a thousand RPM and then is turned off.The tissue at this time is very S eyelets are very sensitive, and you don't want to over compact them. The tubes are removed and we will do this wash step three more times. This is the end of the third wash.The components of the 50 mil conical tube are transferred to the 15 conical tube and centrifuge for a thousand RP M and then stopped. The supernat will be removed and the first part of the FI Call will be started. We are at the step where we Have our pelleted di pancreas digest.We'll resuspend that tissue so it's nice and loose to the pellets. We'll add the first density gradient, which is 1 1 0 8. We'll add five mils to each tube.The pellet is then mixed thoroughly in each of the gradients. It's important that you get good tissue distribution in your heaviest gradient. Now we will layer two mils of 1 0 9 6 density.We will then on top of that, layer two mils of 1 0 6 9 density, and then on top of that 1 0 3 7 density. It's important while you're layering the densities that you don't mix. Then all the layers have been added to the tube.And you can see this is the top of 1 1 0 8, the top of 9 1 0 9 6, the top of 1 0 6 9 and the top of 1 0 3 7. And you could see the tissue is already starting to move to its proper density even without centri fusion. The fial gradients are brought up to speed at 1800 RPM for 15 minutes, and remember to keep the brake off after the tubes are removed from the centrifuge.You can see between the 0 0 9 6 and 0 0 7 layer, there's a nice band of eyelet tissue Pablo's. Now removing the 0 3 7 layer to remove any tissue debris and DNA that might have been released during the processing. Using a sterile plastic pasture pipette, he will now remove the islet layer and transfer them to a 50 mil conical containing 25 mils of wash buffer.You want do this step quickly because fol is toxic to eyelets. We actually removed the entire 0 6 9 and 0 9 6 layer. Pavo cuts the top of the plastic pasture pipette and rinses it with wash buffer.The eyelets are then centrifuge like we centrifuge previously. Centrifuge is brought up to a Thousand RPM and then we stop it and We aspirate off. The supernatant eyelets will be washed three times with wash media.So we just completed the eyelid Isolation process we took from the digest to the fol gradient to the purified eyelets after our third wash with wash buffer, we resuspended the eyelets in our RPMI media and we played it in a tissue culture plate. These eyelets are now ready for in vitro assays or in vivo preps like eyelet Transplant.

Murine Inselzellen der Bauchspeicheldrüse Isolation

Forschung

Biologie

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Einzellige Transkriptomischen Analysen der Maus endokrinen Zellen der Bauchspeicheldrüse

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Entwicklungsbiologie

Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate the measurement of islet oxygen consumption using a 96-well microplate designed for the measurement of oxygen consumption in spheroids. In this assay, spheroid microplates are coated with a cell and tissue adhesive on the day prior to the assay. We utilize a small volume of adhesive solution to encourage islet adherence to only the bottom of the well. On the day of the assay, 15 islets are loaded directly into the base of each well using a technique that ensures optimal positioning of islets and accurate measurement of oxygen consumption. Various aspects of mitochondrial respiration are probed pharmacologically in non-human primate islets, including ATP-dependent respiration, maximal respiration, and proton leak. This method allows for consistent, reproducible results using only a small number of islets per well. It can theoretically be applied to any cultured spheroids of similar size.

This protocol demonstrates the accurate and reproducible measurement of oxygen consumption in non-human primate pancreatic islets. The islet loading techniques and coating of the microplate provide a framework for efficient measurement of respiration in other types of cultured spheroids.

Analyse des Sauerstoffverbrauchs der nichtmenschlichen Primaten-Pankreas-Islet

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate ...

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; Mausmodell des Typ-2-Diabetes: Pancreatic Islet Isolation und Live-cell 2-Photonen-Imaging von intakten Inseln

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, ...

Medizin

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 &#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.

Ein hoher Gehalt<em&gt; In Vitro</em&gt; Pancreatic Islet β-Zell-Replikation-Discovery-Plattform

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

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

Ein In Ovo -Modell zum Testen Insulin-mimetischen Verbindungen

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

Neurowissenschaften

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Intraportal Transplantation der Bauchspeicheldrüse Inselchen im Mausmodell

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Eine Methode zur Maus-Inselzellen der Bauchspeicheldrüse Isolation und intrazellulären cAMP-Bestimmung

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Ein einfaches Hocheffizienzprotokoll für die Isolierung von Pankreas-Isleten von Mäusen

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Quantitative Analyse der Mitophagie in murinen Pankreas-β-Zellen

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in &#946;-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify &#946;-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for &#946;-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in &#946;-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous &#946;-cell models.

Autor im Rampenlicht: Bewertung des Mitophagieflusses in Pankreas-β-Zellen unter Verwendung effektiver und robuster komplementärer Ansätze 

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic &#946;-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

Komplementäre Ansätze zur Untersuchung des Mitophagieflusses in β-Zellen der Bauchspeicheldrüse

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient ...

Ein murinen Pancreatic Islet Cell-basierte Screening für diabetogene Umweltchemikalien

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video