Name: assessing the secretory capacity of pancreatic acinar cells
Uploaded: 2014-08-28T15:00:00
Description: Watch this Scientific Journal Video about Assessing the Secretory Capacity of Pancreatic Acinar Cells at JoVE.com

Dette arbejde blev finansieret af en bevilling fra US-Israel BSF til BS og EDSBS er en etableret af Hilda og Cecil Lewis professoratet i molekylær genetik.

BEMÆRK: procedurer, der involverer dyreindivider er blevet godkendt af Institutional Animal Care og brug Udvalg (IACUC) ved Weizmann Institute of Science. 1. Prøvefremstilling <ol><li> Isolering af pancreas acini <ol><li> Forbered 300 ml frisk Krebs-Ringer bicarbonat HEPES (KRBH), før du begynder. Bland 140 mM NaCI, 4,7 mM KCI, 1,16 mM MgCl2, 1 mM CaCl2, 11 mM glucose, 10 mM HEPES. Tilføj bovint serumalbumin (BSA) og 0,1 mg / ml sojabønnetrypsininhibitor (STI) (r...

<ol>
	<li>Williams, 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=Regulation+of+pancreatic+acinar+cell+function.">Regulation of pancreatic acinar cell function.</a> Current Opinion in Gastroenterology. 22, 498-504 (2006).</li><li>Schnekenburger, 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=The+role+of+kinesin,+dynein+and+microtubules+in+pancreatic+secretion.">The role of kinesin, dynein and microtubules in pancreatic secretion.</a> Cellular and Molecular Life Sciences : CMLS. 66, 2525-2537 (2009).</li><li>Geron, E., Schejter, E. D., Shilo, B. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directing+exocrine+secretory+vesicles+to+the+apical+membrane+by+actin+cables+generated+by+the+formin+mDia1.">Directing exocrine secretory vesicles to the apical membrane by actin cables generated by the formin mDia1.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 10652-10657 (2013).</li><li>Amsterdam, A., Jamieson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+characterization+of+isolated+pancreatic+exocrine+cells.">Structural and functional characterization of isolated pancreatic exocrine cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 69, 3028-3032 (1972).</li><li>Williams, J. A., Korc, M., Dormer, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+secretagogues+on+a+new+preparation+of+functionally+intact,+isolated+pancreatic+acini.">Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini.</a> The American journal of physiology. 235, 517-524 (1978).</li><li>Jerdeva, G. V., 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=Actin+and+non-muscle+myosin+II+facilitate+apical+exocytosis+of+tear+proteins+in+rabbit+lacrimal+acinar+epithelial+cells.">Actin and non-muscle myosin II facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells.</a> Journal of Cell Science. 118, 4797-4812 (2005).</li><li>Menozzi, D., Jensen, R. T., Gardner, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispersed+pancreatic+acinar+cells+and+pancreatic+acini.">Dispersed pancreatic acinar cells and pancreatic acini.</a> Methods in Enzymology. , 192-271 (1990).</li><li>Bi, Y., Williams, 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=A+role+for+Rho+and+Rac+in+secretagogue-induced+amylase+release+by+pancreatic+acini.">A role for Rho and Rac in secretagogue-induced amylase release by pancreatic acini.</a> Am J Physiol Cell Physiol. 289, C22-C32 (2005).</li><li>Palade, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+aspects+of+the+process+of+protein+synthesis.">Intracellular aspects of the process of protein synthesis.</a> Science. 189, 867 (1975).</li><li>Masedunskas, A., 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=Role+for+the+actomyosin+complex+in+regulated+exocytosis+revealed+by+intravital+microscopy.">Role for the actomyosin complex in regulated exocytosis revealed by intravital microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 13552-13557 (2011).</li><li>Petersen, H., Grossman, M. 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Udover ex vivo kultur, alternative opstillinger til studiet af eksokrine sekretion omfatter intravital mikroskopi og måling af væsker fra pankreasgangen. Intravital mikroskopi blev vist sig at fungere godt i mus spytkirtler 10. Denne metode kan optage sekretion i realtid i det intakte dyr, men er begrænset i sin beslutning og midlerne til at manipulere exokrine væv. Vurdering sekretion ved at samle fluider fra pankreasgangen blev opnået i bedøvede rotter 11. Men da kanylering af pank...

Den exokrine pancreas udgør det meste af massen af pattedyr bugspytkirtel og er dedikeret til produktion og sekretion af fordøjelsesenzymer 1. Den funktionelle enhed af eksokrine bugspytkirtlen, acinus, er en klynge af epitelceller, som danner og deler en fælles lumen. Ved hormonal og neuronal stimulation er vesikler fyldt med enzymer, transporteres til den apikale celleoverfladen, sikring med det og uddrive deres indhold ind i hulrummet 1-3. De udskilte enzymer derefter drænes af sammensmeltende sæt af kanaler i tyndtarmen, hvor de katalyserer nedbrydningen af ​​fødevarer i næringsstoffer. Denne video viser, hvordan intakt acini isoleres fra hele bugspytkirtlen, og hvordan deres sekretorisk kapacitet kan vurderes. Ved hjælp af denne opsætning er pancreassekretion kvantificeres ved at måle den relative mængde amylase, der blev udgivet efter stimulering. Alternativt kan sekretion skal afbildes direkte ved anvendelse af forskelligesensorer og farvestoffer. Ex vivo opsætning af pancreas-acini er fordelagtig, eftersom isoleret acini, som bevarer mange karakteristika af de intakte bugspytkirtlen, kan manipuleres og overvåges lettere end i hele dyret 4-6. Denne opsætning blev oprindelig udviklet i slutningen af 1970&#39;erne og var siden forlænget til undersøgelse af flere exokrine væv, såsom spyt og mælkekirtler 3-7. Især det giver studiet af sekretion med minimal indblanding på de komplekse cellulære organisationer i disse polariserede epithel.

<table border="0" cellpadding="0" cellspacing="0" width="655">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">ICR mice</td>
			<td style="width:115px;">Harlan</td>
			<td style="width:91px;"></td>
			<td style="width:203px;">Any strain will do</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HBSS</td>
			<td>Sigma Aldrich</td>
			<td>H8264</td>
			<td>Can substitude for KRB</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Sigma Aldrich</td>
			<td>A7906</td>
			<td>Fraction V</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin inhibitor</td>
			<td>Sigma Aldrich</td>
			<td>T9003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase XI&#160;</td>
			<td>Sigma Aldrich</td>
			<td>C0130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nylon mesh</td>
			<td>BD Falcon</td>
			<td>352360</td>
			<td>70-100&#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amylase infinity reagent&#160;</td>
			<td>Thermo</td>
			<td>TR25421</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CCK</td>
			<td>Sigma Aldrich</td>
			<td>C2175</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FM4-64</td>
			<td>Lifetechnologies</td>
			<td>F34653</td>
			<td>Diluted to 16&#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20mL scintillation vial</td>
			<td>Sigma Aldrich</td>
			<td>Z190527</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DeltaVision system&#160;</td>
			<td>Applied Precision</td>
			<td></td>
			<td>Consisting of an inverted microscope IX71 equipped with 60x/1.4 or 100x/1.3 objectives (Olympus)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss 510 or 710 confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>60x/1.4 or 100x/1.4 objectives&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra Microplate reader</td>
			<td>BioTek</td>
			<td>ELx808</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing the secretory capacity of pancreatic acinar cells

Pancreas acinære celler producerer og udskiller fordøjelsesenzymer. Disse celler er organiseret som en klynge, der danner og deler en fælles lumen. Dette arbejde viser, hvordan sekretorisk kapacitet af disse celler kan vurderes ved dyrkning af isolerede acini. Opsætningen er en fordel, da isolerede acini, som bevarer mange egenskaber intakte exokrin bugspytkirtlen kan manipuleres og overvåges lettere end i hele dyret. Korrekt isolering af pancreas acini er et centralt krav, således at ex vivo kultur vil repræsentere in vivo natur acini. Protokollen viser, hvordan man isolere intakt acini fra muse bugspytkirtlen. Efterfølgende to komplementære metoder til evaluering pancreassekretion fremlagt. Den amylasesekretion analysen fungerer som et globalt mål, mens direkte billeddannelse af pancreassekretion tillader karakterisering af sekretion ved en sub-cellulær opløsning. Kollektivt, de teknikker Presented her muliggøre et bredt spektrum af forsøg for at studere eksokrine sekretion.

Pancreas acini der blev isoleret ordentligt vise en stereotyp morfologi, når de ses ved gennemlysning. Deres basolaterale domæner skal vises rundt og blottet for bleb&#39;er. Den apikale domæne er omgivet af hundredvis af sekretoriske vesikler og synes mørkere i farven (figur 2A, B). Kernerne placeret basal til vesikulær område. Cellerester og komponenter i pancreas gangsystemet og endokrine bugspytkirtlen, som kan detekteres ved tidlige stadier af acini isolering (figur 2C, D) b?...

Watch this Scientific Journal Video about Assessing the Secretory Capacity of Pancreatic Acinar Cells at JoVE.com

Assessing the Secretory Capacity of Pancreatic Acinar Cells

Isolerede pancreas acini bevarer deres in vivo morfologi og aktivitet og tilbyde effektive måder til at overvåge og manipulere sekretion. Dette arbejde viser, hvordan acini kan isoleres fra muse-bugspytkirtlen, og hvordan deres sekretoriske kapacitet kan vurderes.

Vurdering af Sekretorisk Kapacitet bugspytkirtlen acinusceller

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work ...

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