Name: collection protocol for human pancreas
Uploaded: 2012-05-23T12:00:00
Description: Watch this Scientific Journal Video about Collection Protocol for Human Pancreas at JoVE.com

Forfatterne takker donorernes familier og de organudtagning involveret i denne forskning og Maria Martino og Irina Kusmartseva for deres eksperthjælp. Dette arbejde blev finansieret af Juvenile Diabetes Research Foundation (MC-T., MA) til støtte for Netværk for Pankreatisk organdonorer med Diabetes.

1. Set-up <ol><li> Tapekassetter til paraffinblokke manuelt med en blyant, eller automatisk med en kassette printer. Medtag donor identifikator (CaseID), prøvetype, alikvot nummer, og eventuelle yderligere entydig identifikation (sag regneark, <a href="http://jove.com/files/ftp_upload/4039/4039appendix1.doc" target="_blank">bilag 1</a> ). </li><li> Label OLT forme som for kassetter med en permanent markør. Klip 5-10 cm rektangler af aluminiumsfolie. </li><li> Udskriv eller manuelt etiket med permanent markør alle h...

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	<li>Brissova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+human+pancreatic+islet+architecture+and+composition+by+laser+scanning+confocal+microscopy.">Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy.</a> J. Histochem. Cytochem. 53, 1087-1097 (2005).</li><li>Rahier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pancreatic+polypeptide+cells+in+the+human+pancreas:+the+effects+of+age+and+diabetes.">The pancreatic polypeptide cells in the human pancreas: the effects of age and diabetes.</a> J. Clin. Endocrinol. Metab. 56, 441-444 (1983).</li><li>Uchida, T., Takada, T., Ammori, B. J., Suda, K., Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+the+ventral+and+dorsal+pancreas:+a+new+insight+into+anatomy+and+embryonic+development.">Three-dimensional reconstruction of the ventral and dorsal pancreas: a new insight into anatomy and embryonic development.</a> J. Hepatobiliary Pancreat. Surg. 6, 176-180 (1999).</li><li>Wittingen, J., Frey, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+concentration+in+the+head,+body,+tail+and+uncinate+process+of+the+pancreas.">Islet concentration in the head, body, tail and uncinate process of the pancreas.</a> Ann. Surg. 179, 412-414 (1974).</li><li>Rahier, J., Goebbels, R., Henquin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+composition+of+the+human+diabetic+pancreas.">Cellular composition of the human diabetic pancreas.</a> Diabetologia. 24, 366-371 (1983).</li><li>Vaught, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+NCI+perspective+on+creating+sustainable+biospecimen+resources.">An NCI perspective on creating sustainable biospecimen resources.</a> J. Natl. Cancer Inst. Monogr. 42, 1-7 (2011).</li><li>Gianani, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+results+of+screening+of+nondiabetic+organ+donors+for+expression+of+islet+autoantibodies.">Initial results of screening of nondiabetic organ donors for expression of islet autoantibodies.</a> J. Clin. Endocrinol. Metab. 91, 1855-1861 (2006).</li><li>Tauriainen, S., Salmela, K., Rantala, I., Knip, M., Hy&#246;ty, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collecting+high-quality+pancreatic+tissue+for+experimental+study+from+organ+donors+with+signs+of+&#946;-cell+autoimmunity.">Collecting high-quality pancreatic tissue for experimental study from organ donors with signs of &#946;-cell autoimmunity.</a> Diabetes Metab. Res. Rev. 26, 585-592 (2010).</li><li>In't Veld, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+insulitis+in+adult+autoantibody-positive+organ+donors.">Screening for insulitis in adult autoantibody-positive organ donors.</a> Diabetes. 56, 2400-2404 (2007).</li><li>T&#246;ns, H. A., Terpstra, O. T., Bouwman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+human+pancreata+in+perspective+of+the+isolation+of+the+islets+of+langerhans.">Heterogeneity of human pancreata in perspective of the isolation of the islets of langerhans.</a> Transplant Proc. 40, 367-369 (2008).</li></ol>

Målet med denne procedure er at definere en standard bugspytkirtlen behandling, der kan harmoniseres på tværs af flere indsamlingssteder og dermed muligt at sammenligne resultater af forskellige grupper. Dissektionsmetoden er baseret på standard kirurgiske patologi praksis med yderligere skridt for annotation af større bugspytkirtel regioner efterfulgt af intra-regionale underafdelinger. Standardisering af biospecimen forarbejdningsmetoder er forpligtet til at lette indsamlingen af høj kvalitet prøver eksemplific...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Dissektion instrumenter, herunder skalpeller med 22 blade </td><td> Various </td><td> Various </td><td></td></tr><tr><td> Centrifugerørene (15 og 50 ml) </td><td> Fisher Scientific </td><td> 05-539-5, 430828 </td><td> Steril </td></tr><tr><td> Cryorør (1,8 ml) </td><td> VWR Nordamerika </td><td> 89004-300 </td><td> Sterile, stregkoder valgfrie </td></tr><tr><td> RNAlater </td><td> Ambion </td><td> AM7021 </td><td></td></tr><tr><td> Kirurgisk Patologi Ink </td><td></td><td></td><td></td></tr><tr><td> Kassetter </td><td> Mercedes Medicinsk </td><td> CAS ECO109 </td><td></td></tr><tr><td> 10% neutralbufret formalin </td><td> Fisher Scientific </td><td> 23-245-685 </td><td></td></tr><tr><td> OLT frysemedium </td><td> TissueTek </td><td> 4583 </td><td></td></tr><tr><td> Forme </td><td> ThermoScientific </td><td> 58952 </td><td> Forskellige størrelser </td></tr><tr><td> Isopentan </td><td> Fisher Scientific </td><td> O3551-4 </td><td> Oktober køling bad </td></tr><tr><td> Tøris </td><td></td><td></td><td> Oktober køling bad </td></tr><tr><td> Flydende nitrogen i Dewar-kolbe </td><td></td><td></td><td> Snap frysepunktet kryohætteglas </td></tr><tr><td> Celledyrkningsskål (100mm) </td><td> Corning </td><td> 430167 </td><td> Steril </td></tr><tr><td> Hyclone DMEF </td><td> ThermoScientific </td><td> SH30023.01 </td><td> Sterilt, lagre nedkølet, glutamin indeholdt </td></tr><tr> &lt;td&gt; føtalt bovint serum </td><td> Cellgro </td><td> 35 til 010-CV </td><td> Sterile, gemme frosne portioner </td></tr><tr><td> Penicillin / streptomycin / Antifungal </td><td> GIBCO </td><td> 15240-062 </td><td> Sterile, gemme frosne portioner </td></tr><tr><td> Type 9 Paraffin </td><td> Richard-Allan Scientific </td><td> 8337 </td><td></td></tr><tr><td> 1X D-PBS </td><td> GIBCO </td><td> 14190-144 </td><td> Steril </td></tr><tr><td> 16% Paraformaldehyd </td><td> EM Sciences </td><td> 15710 </td><td> Gør frisk, Store kølet </td></tr><tr><td> 8% glutaraldehyd </td><td> EM Sciences </td><td> 16019 </td><td> Gør frisk, Store kølet </td></tr><tr><td> Biosikkerhedsniveau 2 hætte </td><td> Various </td><td></td><td></td></tr><tr><td> Ultra-lav fryser </td><td> Various </td><td></td><td></td></tr><tr><td> Sakura kassette Printer </td><td> Sakura </td><td></td><td></td></tr><tr><td> Sakura VIP300 </td><td> Sakura </td><td></td><td> Automatisk paraffin processor </td></tr><tr><td> Paraffin Embedding Station </td><td> ThermoFisher </td><td></td><td></td></tr></tbody></table> Tabel 1. Specifikke reagenser og udstyr.

collection protocol for human pancreas

Dette dissektion og prøveudtagningsprocedure blev udviklet for Netværk for Pankreatisk organdonorer med Diabetes (nPOD) program til at standardisere forberedelse af bugspytkirtlen genvundet fra døde organdonorer. Bugspytkirtlen er opdelt i 3 vigtigste regioner (hoved, krop, hale), efterfulgt af serielle tværsnit i hele medialt for laterale akse. Skiftevise sektioner anvendes til fast paraffin og friske frosne blokke og tilbageværende prøver hakkes til lynfrosset prøve præparater, enten med eller uden RNAse inhibitorer, DNA, RNA eller protein isoleret. Det overordnede mål i bugspytkirtlen dissektion procedure er at prøve det hele bugspytkirtlen og samtidig opretholde anatomiske orientering. Endokrine celle heterogenitet i form af ø-sammensætning, størrelse og tal er rapporteret for humane øer i forhold til gnaver holme 1. Størstedelen af ​​humane øer fra bugspytkirtlen hovedet er krop og hale regioner er sammensat af insulin-containing p-celler efterfulgt af lavere andele af glucagon-indeholdende a-celler og somatostatin-holdige △-celler. Pankreatiske polypeptid-holdige PP-celler og ghrelin-holdige epsilon celler er også til stede, men i mindre mængder. I modsætning hertil indeholder uncinate region øer, der primært består af pankreatiske polypeptid-holdige PP-celler 2. Disse regionale ø variationer opstår udviklingsmæssige forskelle. Bugspytkirtlen udvikler sig fra de ventrale og dorsale pancreas knopper i formave og efter rotation i maven og duodenum de ventrale lap træk og sikringer med den dorsale 3. Den ventrale lap danner den bageste del af hovedet, herunder uncinate processen, mens dorsal lap giver anledning til resten af ​​organet. Regional bugspytkirtlen variation er også rapporteret med halen regionen har højere holm massefylde sammenlignet med andre regioner, og den dorsale Lap-afledte komponenter undergår selektiv atrofi i type 1-diabetes <sop&gt; 4,5. Yderligere organer og væv er ofte inddrives fra organdonorer og omfatter pancreas lymfeknuder, milt og ikke-pancreas lymfeknuder. Disse prøver udvundet med samme format som for pancreas med tilsætning af isolering af kryopræserverede celler. Når den proksimale duodenum er inkluderet i bugspytkirtlen, kan duodenal mucosa skal indsamles til paraffin og frosne blokke og hakket snap frosne præparater.

Watch this Scientific Journal Video about Collection Protocol for Human Pancreas at JoVE.com

Collection Protocol for Human Pancreas

Denne video viser en dissektion procedure for behandling af human pancreas i flere lagringsformater. Anatomisk orientering opretholdes under hele pancreas regioner tillader definition af regional ø sammensætning og densitet.

Indsamling Protokol for human pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

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Medicine

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