मानव अग्नाशय के लिए संग्रह प्रोटोकॉल
Summary
इस वीडियो एकाधिक भंडारण स्वरूपों में मानव अग्न्याशय के प्रसंस्करण के लिए एक विच्छेदन प्रक्रिया को दर्शाता है. क्षेत्रीय आइलेट संरचना और घनत्व की परिभाषा की अनुमति संरचनात्मक अभिविन्यास अग्नाशय क्षेत्रों भर में बनाए रखा है.
Abstract
This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing β-cells followed by lower proportions of glucagon-containing α-cells and somatostatin-containing δ-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.
Protocol
Discussion
इस प्रक्रिया के लक्ष्य के लिए एक मानक अग्न्याशय प्रसंस्करण है कि कई संग्रह साइटों के पार संगत हो सकता है परिभाषित करने और इस तरह अलग अलग समूहों द्वारा प्राप्त परिणामों की तुलना की अनुमति है. विच्छेदन व…
Disclosures
The authors have nothing to disclose.
Acknowledgements
लेखकों 'दाताओं के परिवारों और अंग प्रत्यारोपण खरीद में शामिल उनके विशेषज्ञ की सहायता के लिए इस शोध और मारिया मार्टिनो और इरीना Kusmartseva के संगठन धन्यवाद. यह काम अग्नाशय के मधुमेह के साथ अंग दाताओं के लिए नेटवर्क के समर्थन में किशोर मधुमेह अनुसंधान फाउंडेशन (MC-T., एमए) द्वारा वित्त पोषित किया गया था.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| Dissection instruments including scalpels with 22 blades | Various | Various | |
| Centrifuge tubes (15 and 50 ml) | Fisher Scientific | 05-539-5, 430828 | Sterile |
| Cryotubes(1.8 ml) | VWR North America | 89004-300 | Sterile, bar codes optional |
| RNAlater | Ambion | AM7021 | |
| Surgical Pathology Ink | |||
| Cassettes | Mercedes Medical | CAS ECO109 | |
| 10% Neutral Buffered Formalin | Fisher Scientific | 23-245-685 | |
| OCT Freezing Media | TissueTek | 4583 | |
| Molds | ThermoScientific | 58952 | Various sizes |
| Isopentane | Fisher Scientific | O3551-4 | OCT chilling bath |
| Dry ice | OCT chilling bath | ||
| Liquid nitrogen in Dewar flask | Snap freezing cryovials | ||
| Cell culture dish (100mm) | Corning | 430167 | Sterile |
| Hyclone DMEF | ThermoScientific | SH30023.01 | Sterile, store refrigerated, glutamine included |
| Fetal Bovine Serum | Cellgro | 35-010-CV | Sterile, store frozen aliquots |
| Penicillin/Streptomycin/Antifungal | GIBCO | 15240-062 | Sterile, store frozen aliquots |
| Type 9 Paraffin | Richard-Allan Scientific | 8337 | |
| 1X D-PBS | GIBCO | 14190-144 | Sterile |
| 16% Paraformaldehyde | EM Sciences | 15710 | Make fresh, store refrigerated |
| 8% Glutaraldehyde | EM Sciences | 16019 | Make fresh, store refrigerated |
| Biosafety Level 2 hood | Various | ||
| Ultra-low freezer | Various | ||
| Sakura Cassette Printer | Sakura | ||
| Sakura VIP300 | Sakura | Automatic paraffin processor | |
| Paraffin Embedding Station | ThermoFisher |
Table 1. Specific reagents and equipment.
References
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