Diese Arbeit wurde vom National Institutes of Health Zuschuss R01DK080971 und Juvenile Diabetes Research Foundation Grant 37-2009-44 unterstützt. Wir schätzen auch Unterstützung von der UCSD Pediatric Diabetes Research Center (PDRC) übermittelt.

1. Transfektion von HEK293 Schicht <ol><li> Bereiten HEK293 Zellmedium durch Zugabe zu 500 ml Flaschen DMEM (mit 4,5 g / ml Glucose und Phenolrot und ohne Glutamin): 50 ml FBS, 5 ml 100X Penicillin / Streptomycin-Lösung, 5 ml 100x L-Glutamin-Lösung und 500 ul Amphotericin B. </li><li> Platte aus HEK293-Zellen in einer 24-Well-Platte unter Verwendung von 0,5 ml pro Vertiefung HEK293 Medien. Stellen Sie sicher, dass die Zellen gleichmäßig auf die Unterseite der Platte verteilt. </li><li> Wenn HEK293-Zellen 100% Konfluenz erreichen Transfektion mit 0,8 ug Plasmid kodiert für das Protein von Interesse (eingesetzt in einem Säugetier-Expressionsvektor) und 2 ul Lipofectamin 2000 gemäß der Lipofectamin-Protokoll. Wir haben entschieden, den pcDNA 3.3 Rückgrat für die Expression in Säugerzellen zu verwenden. </li><li> Optional kann nach 6 h Inkubation Austausch die Transfektion Medien beschrieben in der Lipofectamine Protokoll mit normalen HEK293 Medien. </li><li> Um die Expression des transfizierten Prote bewertenin, kann ein Teil der transfizierten Gewebekulturvertiefungen für Immunfluoreszenz Nachweis des exprimierten Proteins oder Western-Blot-Analyse von Protein-Expression unter Verwendung von Standard-Techniken verwendet werden. </li></ol> 2. Optional Fixation von HEK293-Zellen transfizierten Protein HEK293 Zellen schonend fixiert werden, um Kokultur in Medien, die schädlich für lebende HEK293 Zellen (z. B. Schritt 3.9 unten) oder um die effiziente Vorbereitung im Vorfeld von Platten für mehrere Experimente auf einmal erlauben (siehe auch Diskussion) erleichtern könnten. <ol><li> Führen Pilotstudien um den zeitlichen Verlauf der Expression des Proteins in den HEK293-Zellen zu bestimmen. </li><li> Nach Transfektion der HEK293 Zellschicht, saugen die Medien aus den Zellen bei der Post-Transfektion Zeitpunkt mit höchster Expression assoziiert. Wenn dies geschieht innerhalb der ersten 24 Stunden, nicht bis 24 Stunden nach der Transfektion anzusaugen. </li><li> Waschen HEK293 Zellen vorsichtig mit D-PBS fügen Sie dann 500 ul 4% Paraformaldehyd (PFA) und Inkubation bei RT für 30 min. (Zu 4% PFA-Lösung zu machen, mit 16% igen Lösung in 1X PBS beginnen und verdünnen 1:4 1X PBS.) </li><li> Mit einer sterilen PBS, vorsichtig waschen die Zellen 3 mal in PBS und Inkubation O / N bei 4 ° C. </li><li> Waschen der Zellen 3 mal mit sterilem PBS, dann fügen PBS und lassen bei 4 ° C bis zur Verwendung (maximale Lagerzeit je nach Protein exprimiert. Wir speichern HEK293 Zellen transfiziert, um neuroligin Isoformen für bis zu 2 Wochen ausdrücken, ohne wesentliche Änderungen in beobachteten Effekte auf die Insulinsekretion). </li></ol> 3. Co-Kultivierung von INS-1 Beta-Zellen mit HEK293 Zellen <ol><li> Bereiten INS-1-Medien durch Zugabe von 500 ml RPMI 1640 (mit Glucose und Phenolrot und ohne Glutamin): 50 ml FBS, 5 ml 100X Penicillin / Streptomycin, 5 ml 100X L-Glutamin-Lösung, 5 ml Natriumpyruvat, 500 ul Amphotericin B, 500 ul beta-Mercaptoethanol (dies ist fast identisch mit dem medium normalerweise für primäre Kultur Insel mit Ausnahme der Zugabe von beta-Mercaptoethanol) eingesetzt. </li><li> Benutzen Sie die Cell-Stripperin (oder einem anderen nicht-enzymatischen Zelle Entferner), Ernte INS-1-Zellen bei etwa 70-80% Konfluenz aus dem Boden einer T75 Kulturflasche. Jeder Kolben wird grob genug Zellen für 48 Brunnen liefern. </li><li> Nach Stillstand Zellen in der Zentrifuge für 3 min bei 1200 rpm in einer 50/50 Mischung aus INS-1 und HEK Medien resuspendieren. Für eine 70-80% konfluenten Flasche INS-1-Zellen, in etwa 25 ml Mischtechnik resuspendieren. </li><li> Bei Verwendung von HEK293-Zellen, die in Paraformaldehyd wurden anstelle von lebenden HEK293 Zellen fixiert, resuspendieren INS-1-Zellen in 100% INS-1-Media anstatt Mischtechnik. </li><li> Verwendung einer 10 ml Pipette zu entfernen, die Zellen aus dem Pellet, um eine homogene Zellsuspension zu machen. </li><li> Saugen Sie die Medien auf den HEK293 Zellen zu entfernen. </li><li> Mit einer Pipette p1000, sanft hinzufügen 500 ul von INS-1 Zell-Suspension auf die HEK Zellschicht an den Seiten of den Brunnen. Nach jeweils 6 Vertiefungen, verwenden Sie eine 10 ml Pipette, um die INS-1-Zellen wieder suspendieren, um eine homogene Zellsuspension zu gewährleisten. Die allgemeine Regelung der Kokultur Set-up ist in Abbildung 1 dargestellt. </li><li> Inkubieren Sie die Zellen für 24-48 h, je nach dem experimentellen Protokoll. </li><li> Wenn mehr als 48 Stunden Kokultur Zeitraum erforderlich ist, verwenden optionale Schritte in Abschnitt 2 die HEK293 Zellen zu fixieren. </li><li> Bei der Verwendung von primären dissoziiert Nagetier oder menschlichen Inselchen [siehe zB vor Protokolle in JoVE 11-15], sicherzustellen, dass eine geeignete Medien wie RPMI mit 5 ml Penicillin / Streptomycin, 5 ml L-Glut, 0,5 ml Amphotericin-B wird anstelle von INS-1-Medien. Obwohl eine dissoziierte Inselzellen in einer Platte mit 24 Vertiefungen verwendet werden können, in der Praxis wegen der großen Anzahl von Inseln möglicherweise benötigt, ist es empfehlenswert, dass die Prüfung der Skalierung gegeben werden auf eine 48-Well-Platte (erfordern weniger Inselzellen pro Vertiefung). Auf einer 48-Well-Platte, fügen Sie etwa 100 Inseln im Wert von Dissoziationted Zellen pro Platte zu starten. Je nach Wirksamkeit der Dissoziation und das Verfahren verwendet wird, kann weniger Inseln erforderlich. </li></ol> 4. Glucose Insulinsekretion <ol><li> Vorinkubieren Zellen in 250 ul 2,5 mM Glukose in KRB (Krebs-Ringer Bicarbonat-Puffer) für mindestens 1 Stunde. Um vorinkubieren, entfernen Kokultur mittel-und unmittelbar mit KRB ersetzen. Dieser Austausch sollte man gut gemacht werden zu einem Zeitpunkt, um die Exposition der INS-1-Zellen zu Umweltinformationen Sauerstoff zu minimieren. Saugen Sie mit einer Hand, während Pipettieren der niedrig (2,5 mM) Glucose KRB mit den anderen. </li><li> Austausch Vorinkubation KRB bis 250 ul von entweder 2,5 mM oder 20 mM Glukose in KRB-Puffer ein und zu einem Zeitpunkt. </li><li> Wenn die für den spezifischen Experiment können zusätzliche secretagogues wie 100 uM IBMX zugegeben, um eine robustere stimulierte Insulinsekretion Reaktion zu erzeugen. Dissoziierte primären Beta-Inselzellen insbesondere sind schlecht auf erhöhte Glucosekonzentrationen Alone, so hier besonders Zugabe von IBMX oder andere Sekretagoga sollte 16,17 in Betracht gezogen werden. </li><li> Nach 1 Stunde Inkubation übertragen Medien in Röhrchen zentrifugieren und Spin 1.500 xg für 5 min. </li><li> Entfernen 200 ul des Überstandes und nutzen diese für die Analyse durch Insulin RIA oder ELISA. </li><li> Um Zelllysat herzustellen, werden 100 ul RIPA Lysepuffer (150 mM NaCl, 1,0% IGEPALCA-630 oder Nonidet P-40, 0,5% Natriumdesoxycholat, 0,1% SDS, 50 mM Tris, pH 8,0) mit Proteaseinhibitoren der Platte sofort nach dem Entfernen von Medien und Inkubation bei 4 ° C für 20 min. </li><li> Spin down Lysat für 15 min bei 10.000 xg und entfernen 50 ul Überstand zur Analyse durch Insulin RIA oder ELISA. </li></ol>

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Wir haben nichts zu offenbaren.

Die Kokultur hier beschriebene Verfahren bietet einen effektiven Weg, um die physiologische Bedeutung der spezifischen Beta-Zell-Oberfläche, Transmembranproteine ​​bestimmen und speziell ihrer extrazellulären Domänen. Durch Kultivierung Betazellen oder Insulinomazellen (wie die INS-1-Zellen verwendet hier) in Kontakt mit HEK293 Zellen, die ein Protein von Interesse auf der Zelloberfläche kann Experimente entwickelt, um die Auswirkungen der extrazellulären Protein-Protein-Wechselwirkungen stören, ohne direkt zu...

Wir beschreiben hier eine Methode, um Untersuchungen, wie die extrazellulären Domänen spezifischer Transmembranproteine ​​beeinflussen Insulinsekretion zu erleichtern. Die Methode Sonden die Auswirkungen der Interaktionen des Proteins von Interesse mit Proteinen (oder möglicherweise andere Moleküle) auf der pankreatischen beta-Zelloberfläche. Das Verfahren ermöglicht Untersuchungen, wie Zelloberflächen-Proteine ​​durch Beta-Zellen oder anderen benachbarten Zellen (zB endothelialen Zellen, Neuronen, Bauchspeicheldrüsen-alpha-Zellen) exprimiert beeinflussen Betazellfunktion durch transzellulären Wechselwirkungen (zB durch Wechselwirkung mit Interaktionspartner auf der Oberfläche des benachbarten Beta-Zellen). Die zelluläre Plasmamembran enthält eine komplexe Anordnung von struktureller und funktioneller Proteine, die als Brücken an die extrazelluläre Umgebung. Durch Bildung von transzellulären Verbindungen oder durch Einleitung von Kunststoff-Signalwege können Wechselwirkungen zwischen Zelloberflächen-Proteine ​​helfen, koordinieren dieFunktion von benachbarten Zellen. Betazellen des Pankreas sind zusammen in den Langerhans-Inseln gruppierten und handeln in einer koordinierten Art und Weise, um Glukose-Homöostase 1 aufrecht zu erhalten. Wie soeben bekannt wurde, zum Beispiel durch die Bedeutung der extrazellulären EphA-EphrinA und neuroligin-2-Wechselwirkungen in der Regulation des Glukose-stimulierte Insulinsekretion, wird es immer klarer, dass eine erhöhte Kenntnis der auftretenden Wechselwirkungen zwischen extrazellulären Proteine ​​auf den Oberflächen von benachbarten Beta-Zellen wird von großer Bedeutung für die Gewinnung ein umfassendes Verständnis der Insulinsekretion der Beta-Zellen funktionelle Reifung und der Aufrechterhaltung der Glukose-Homöostase 1-3 sein. Das Ziel der hier beschriebenen Methode ist es, Untersuchungen der Auswirkungen auf die Beta-Zell-Funktion transzellulären Wechselwirkungen mit spezifischen Transmembran oder anderweitig-Zell-Oberflächen-assoziierten Proteinen ermöglichen. Durch Co-Kultivierung Beta-Zellen mit HEK293 Zellen mit unterschiedlichen Ausdruck Konstrukten transfiziert, die Auswirkungen auf die beta-Zell-Funktion verschiedener Zelloberflächen-Proteine ​​oder mutierten Varianten davon können effizient sondiert. Dies wird ohne transfektieren die Beta-Zellen selbst zu erreichen. Aufklärung der Rolle der insbesondere transzellulären Wechselwirkungen durch Knockdown, Knockout oder Überexpression Studien in kultivierten Beta-Zellen oder in vivo erfordert direkte Störung der Beta-Zell-mRNA und Protein-Expression, möglicherweise beeinflussen Betazellen Gesundheit und / oder die Funktion in einer Weise, die Analysen verwechseln könnte die Auswirkungen der extrazellulären Wechselwirkungen. Diese nähert sich auch ändern Niveaus der intrazellulären Domänen der Zielproteine ​​und ferner nicht erlauben Effekte aufgrund von Wechselwirkungen zwischen Proteinen, die am oder in der gleichen Zelle unter den Auswirkungen von Wechselwirkungen transzellulären unterscheiden. Hier ist ein Verfahren zur Bestimmung der Wirkung von spezifischen transzellulären Wechselwirkungen auf die Insulin-sezernierenden Kapazität und Reaktionsfähigkeit der Betazellen Artbezibed. Dieses Verfahren ist für insulinproduzierenden ß-Zelllinien, wie INS-1-Zellen 4 und dissoziierte primären Nagetier oder humanen beta-Zellen. Es basiert auf Kokultur Modelle von Neurobiologen, die, dass die Exposition von kultivierten Neuronen spezifische neuronale Proteine ​​auf HEK293 (oder COS) Zellschichten exprimiert gefunden entwickelten Proteine ​​identifizieren konnte, die Fahrt Synapsenbildung 5,6. Angesichts der Parallelen zwischen der sekretorischen Maschinerie der neuronalen Synapsen und der Beta-Zellen, begründeten wir, dass Beta-Zell-Funktion und funktionelle Reifung durch ähnliche transzellulären Wechselwirkungen 7-9 könnte angetrieben werden. Um diese Wechselwirkungen untersuchen, haben wir das hier beschriebene System in der Beta-Zellen auf einer Schicht aus HEK293 Zellen, die ein Protein von Interesse 10 sind co-kultiviert. Dieses System ermöglicht die Beta-Zell-Zytoplasma unangetastet bleiben, während extrazelluläre Protein-Protein-Wechselwirkungen manipuliert werden.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>pcDNA 3.3 vector/backbone</td>
			<td>Invitrogen</td>
			<td>K830001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>18324012</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Mediatech</td>
			<td>45001-312</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen/strep solution</td>
			<td>Mediatech</td>
			<td>45001-652-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Mediatech</td>
			<td>45001-808-1/30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Mediatech</td>
			<td>45001-404</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Mediatech</td>
			<td>45001-434</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Mediatech</td>
			<td>45001-710-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985023</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell stripper</td>
			<td>Mediatech</td>
			<td>45000-668</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>T75 Flask</td>
			<td>BD</td>
			<td>1368065</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>50980487</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Mediatech</td>
			<td>45001-130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Mediatech</td>
			<td>MT35010CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Sigma</td>
			<td>I5879-100MG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RIPA lysis buffer</td>
			<td>Sigma</td>
			<td>R0278</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

coculture analysis of extracellular protein interactions affecting insulin secretion by pancreatic beta cells

Wechselwirkungen zwischen Zelloberflächen-Proteine ​​zur Koordination der Funktion der benachbarten Zellen. Betazellen der Bauchspeicheldrüse zusammen innerhalb Pankreasinseln gruppierten und handeln in einer koordinierten Art und Weise, um Glukose-Homöostase aufrecht zu erhalten. Es wird zunehmend deutlich, dass Wechselwirkungen zwischen Transmembranproteine ​​auf den Oberflächen der angrenzenden Beta-Zellen wichtige Determinanten der Beta-Zell-Funktion sind. Aufklärung der Rolle der insbesondere transzellulären Wechselwirkungen durch Knockdown, Knockout oder Überexpression Studien in kultivierten Beta-Zellen oder in vivo erfordert direkte Störung der mRNA und Protein-Expression, möglicherweise beeinflussen beta-Zelle Gesundheit und / oder die Funktion in einer Weise, die Analysen über die Auswirkungen verwechseln könnte von spezifischen Wechselwirkungen. Diese Ansätze auch ändern Niveaus der intrazellulären Domänen der Zielproteine ​​und kann Auswirkungen auf Wechselwirkungen zwischen Proteinen innerhalb der gleichen Zelle Membran zu verhindern, dass disunterschieden von den Auswirkungen der transzellulären Wechselwirkungen. Hier wird ein Verfahren zur Bestimmung der Wirkung von spezifischen transzellulären Wechselwirkungen auf die Insulin-sezernierenden Kapazität und Reaktionsfähigkeit der Beta-Zellen präsentiert wird. Dieses Verfahren ist für Beta-Zell-Linien, wie INS-1-Zellen, und distanzierte primären Beta-Zellen. Es basiert auf Kokultur Modelle von Neurobiologen, die, dass die Exposition von kultivierten Neuronen spezifische neuronale Proteine ​​auf HEK293 (oder COS) Zellschichten identifizierten Proteine ​​wichtig für den Antrieb Synapsenbildung Ausdruck gefunden entwickelt wurde. Angesichts der Parallelen zwischen der sekretorischen Maschinerie der neuronalen Synapsen und der Beta-Zellen, begründeten wir, dass beta-Zelle funktionelle Reifung durch ähnliche transzellulären Wechselwirkungen könnte angetrieben werden. Wir entwickelten ein System, in dem Beta-Zellen auf einer Schicht aus HEK293 Zellen, die ein Protein von Interesse kultiviert werden. In diesem Modell wird das Beta-Zell-Zytoplasma unberührt, während extrazellulären Protein-Protein interactions manipuliert werden. Obwohl wir hier in erster Linie auf Studien von Glukose-stimulierte Insulinsekretion zu konzentrieren, können andere Prozesse analysiert werden, z. B. Veränderungen in der Genexpression durch Immunoblot oder qPCR bestimmt.

Mit dem hier beschriebenen Verfahren haben wir die Wirkung von verschiedenen Varianten des Proteins neuroligin auf die Insulinsekretion getestet. Dies ergänzt unsere veröffentlichte Arbeit untersucht die Wirkung von neuroligin-2 auf Beta-Zell-Funktion 10. Abbildung 2, zum Beispiel, stellt Ergebnisse von Cokultivierung INS-1 beta-Zellen mit HEK293-Zellen transfiziert, um einen Ausdruck zu erhalten neuroligin Isoform hier bezeichnet als NL- X. Dieses Experiment wurde entworfen, um die Hypothe...

Watch this Scientific Journal Video about Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells at JoVE.com

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Transzelluläre Protein-Interaktionen sind wichtige Determinanten der Pankreas-Beta-Zell-Funktion. Hier ist eine Methode der detaillierten angepasste aus einer Co-Kultur-Modell-Synaptogenese zur Untersuchung, wie bestimmte Transmembranproteine ​​Insulinsekretion beeinflussen. HEK293 Zellen exprimieren Proteine ​​von Interesse; Beta-Zellen brauchen nicht transfiziert oder anderweitig direkt gestört.

Cokultur Analyse der extrazellulären Protein Wechselwirkungen, die Insulinsekretion durch Betazellen des Pankreas

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

coculture-analysis-extracellular-protein-interactions-affecting

Research

JoVE Journal

Medicine

12.8K Aufrufe.  University of California, San Diego. Transzelluläre Protein-Interaktionen sind wichtige Determinanten der Pankreas-Beta-Zell-Funktion. Hier ist eine Methode der detaillierten angepasste aus einer Co-Kultur-Modell-Synaptogenese zur Untersuchung, wie bestimmte Transmembranproteine ​​Insulinsekretion beeinflussen. HEK293 Zellen exprimieren Proteine ​​von Interesse; Beta-Zellen brauchen nicht transfiziert oder anderweitig direkt gestört.

Serie: Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Improving IV Insulin Administration in a Community Hospital

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose concentrations, even in non-diabetic patients, predicts poor outcomes.1-4 The 2008 consensus statement by the American Association of Clinical Endocrinologists (AACE) and the American Diabetes Association (ADA) states that "hyperglycemia in hospitalized patients, irrespective of its cause, is unequivocally associated with adverse outcomes."5 It is important to recognize that hyperglycemia occurs in patients with known or undiagnosed diabetes as well as during acute illness in those with previously normal glucose tolerance.
The Normoglycemia in Intensive Care Evaluation-Survival Using Glucose Algorithm Regulation (NICE-SUGAR) study involved over six thousand adult intensive care unit (ICU) patients who were randomized to intensive glucose control or conventional glucose control.6 Surprisingly, this trial found that intensive glucose control increased the risk of mortality by 14% (odds ratio, 1.14; p=0.02). In addition, there was an increased prevalence of severe hypoglycemia in the intensive control group compared with the conventional control group (6.8% vs. 0.5%, respectively; p&lt;0.001). From this pivotal trial and two others,7,8 Wyoming Medical Center (WMC) realized the importance of controlling hyperglycemia in the hospitalized patient while avoiding the negative impact of resultant hypoglycemia. 
Despite multiple revisions of an IV insulin paper protocol, analysis of data from usage of the paper protocol at WMC shows that in terms of achieving normoglycemia while minimizing hypoglycemia, results were suboptimal. Therefore, through a systematical implementation plan, monitoring of patient blood glucose levels was switched from using a paper IV insulin protocol to a computerized glucose management system. By comparing blood glucose levels using the paper protocol to that of the computerized system, it was determined, that overall, the computerized glucose management system resulted in more rapid and tighter glucose control than the traditional paper protocol. Specifically, a substantial increase in the time spent within the target blood glucose concentration range, as well as a decrease in the prevalence of severe hypoglycemia (BG &lt; 40 mg/dL), clinical hypoglycemia (BG &lt; 70 mg/dL), and hyperglycemia (BG &gt; 180 mg/dL), was witnessed in the first five months after implementation of the computerized glucose management system. The computerized system achieved target concentrations in greater than 75% of all readings while minimizing the risk of hypoglycemia. The prevalence of hypoglycemia (BG &lt; 70 mg/dL) with the use of the computer glucose management system was well under 1%.

When compared to the previous paper protocol, implementation of a computerized glucose management system results in a substantial increase in blood glucose concentration measurements within the target range. Using a computerized glucose management system to monitor blood glucose levels, decreases in severe hypoglycemia (&lt;40 mg/dL), clinical hypoglycemia (&lt;70 mg/dL), and hyperglycemia (&gt;180 mg/dL) also can be observed.

Die Verbesserung der IV-Verabreichung von Insulin in einem Community Hospital

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose ...

Forschung

Medizin

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Isolation, Charakterisierung und vergleichende Differenzierung humaner Dental Pulp Stammzellen aus bleibenden Zähne mit zwei verschiedenen Methoden abgeleitet

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Co-Kultur-System mit einer Scheibe Organotypische Gehirn-und 3D-Sphäroid von Karzinomzellen

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

Human Dupuytren&#039;s Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Dupuytren&#8217;s disease (DD) is a fibroproliferative disease of the palm of the hand. Here, we present a protocol to culture resection specimens from DD in a three-dimensional (3D) culture system. Such short-term culture system allows preservation of the 3D structure and molecular properties of the fibrotic tissue.

Menschen Dupuytren&#39;sche<em&gt; Ex vivo</em&gt; Kultur für das Studium der Myofibroblasten und extrazellulären Matrix-Interaktionen

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Studieren Bauchspeicheldrüsenkrebs Stammzelleigenschaften für die Entwicklung neuer Behandlungsstrategien

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (A&#946;o) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble A&#946;o correlate with memory deficits in AD models and humans. The A&#946;o-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated A&#946;o infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning A&#946;o-induced memory deficits at the onset of AD. Since soluble A&#946;o aggregate relatively fast into insoluble A&#946; fibrils that correlate poorly with the clinical state of patients, soluble A&#946;o are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of A&#946;o and continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of A&#946;o in pre-dementia patients.

To study the effects of A&#946;o in vivo, we developed a model based on repeated hippocampal infusions of soluble A&#946;o coupled with continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of A&#946;o.

Neurodegenerative Erkrankungen im Tiermodell der chronischen Amyloid-beta-Oligomers Infusion wird durch Antikörper-Behandlung Angereichert mit osmotische Pumpen entgegen gewirkt

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It ...

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.

Quantitative 3D<em&gt; In Silico</em&gt; Modellierung (q3DISM) zerebraler Amyloid-beta Phagozytose in Nagetiermodellen der Alzheimer-Krankheit

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

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved F&#246;rster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved F&#246;rster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Homogeneous Time-resolved F&amp;#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

Homogene Zeitaufgelösten Förster Resonanz Energie Transfer-basierten Test zur Erkennung von Insulin-Sekretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

Isolierung der primäre Maustaste Hepatozyten für entstehende Protein Synthese Analyse von nicht-radioaktiven L-Azidohomoalanine Kennzeichnung Methode

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

Bio-Energetik-Untersuchung von Candida Albicans mittels Real-Time extrazelluläre Flux Analyse

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...