人体胰腺的收集协议
Summary
该视频演示了加工成多种存储格式的人类胰腺的解剖过程。解剖方向，保持整个胰腺地区允许区域胰岛成分和密度的定义。
Abstract
这解剖和取样程序开发为糖尿病（nPOD）的计划，以规范准备恢复从尸体器官捐献者的胰腺胰腺器官捐赠者的网络。胰腺分为3个主要地区整个横向轴内侧串行的横截面（头，身，尾）。交替部分用于固定石蜡和鲜冻块和残留样品剁碎单元冷冻样品准备，无论有或无RNase抑制剂，DNA，RNA或蛋白质隔离。胰腺的解剖过程的总体目标是保持解剖定位的同时，品尝整个胰腺。
在小岛组成，大小和数字方面的内分泌细胞的异质性是人类胰岛的报告相比，啮齿动物胰岛1。大多数人类胰岛胰头，身体和尾巴地区组成的胰岛素containinGβ-细胞的比例较低，其次是胰高血糖素的α细胞和生长抑素的δ-细胞。胰多肽含PP细胞生长素含小量细胞也存在，但在小数字。相比之下，钩突地区包含主要胰岛胰多肽含PP细胞组成。这些区域的胰岛变化，从发展的差异产生。胰腺发展的前肠和胃和十二指肠，腹侧叶动作和保险丝背3旋转后的腹侧和背侧胰芽。腹侧叶形成的头后部，包括钩突背叶而产生的其他器官。另据报道，尾部区域，具有较高的胰岛细胞密度比其他地区和背叶源性成分，在1型糖尿病的发生选择性萎缩区域胰腺癌的变化<s高达> 4,5。
其他器官和组织往往是恢复从器官捐赠者和包括胰腺淋巴结，脾脏和非胰腺淋巴结。这些样品被收回冻存细胞的分离此外胰腺类似的格式。当近端十二指肠与胰腺，十二指肠粘膜可收集石蜡和冰冻块和碎单元冷冻的筹备工作。
Protocol
1。设置石蜡块，用铅笔或自动录像带打印机手动标签的录像带。包括捐赠标识符（CaseID），样本类型，等分数目，以及任何额外的唯一标识（案例表， 附录1 ）。 标签华侨城模具为永久性标记录音带。剪下铝箔5-10厘米的长方形。 打印或手工标签永久性标记所有小瓶和管。包括足够瓶碎单元冷冻样品和新鲜出?…
Discussion
此过程的目标是定义一个标准的胰腺处理，可以跨多个收集点统一，从而使不同群体得到的结果进行比较。剥离的方法是基于标准的注释主要胰腺区域内细分区域的额外步骤与手术病理做法。标准化的biospecimen处理方法是必需的，以便收集高品质的最佳做法biospecimen收集出版由国家组织，如国家癌症研究所6 biobanking体现样品。从捐助者代表糖尿病和适当的控制人口的各个阶段的人类胰腺样?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者感谢捐助者的家庭和组织的专家协助参与这项研究和玛丽亚·马蒂诺和伊琳娜Kusmartseva器官采购。这项工作是由青少年糖尿病研究基金会（MC-T.，马萨诸塞州）在对糖尿病患者的胰腺器官捐赠者的网络支持。
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.
Referências
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Tags
Collection ProtocolHuman PancreasDissectionSampling ProcedureNetwork For Pancreatic Organ Donors With Diabetes (nPOD)StandardizeCadaveric Organ DonorsPancreas RegionsHeadBodyTailSerial Transverse SectionsFixed ParaffinFresh Frozen BlocksSnap Frozen Sample PreparationsRNAse InhibitorsDNARNAProtein IsolationPancreas Dissection ProcedureAnatomical OrientationEndocrine Cell HeterogeneityIslet CompositionSizeNumbersInsulin-containing β-cellsGlucagon-containing α-cellsSomatostatin-containing δ-cellsPancreatic Polypeptide-containing PP CellsGhrelin-containing Epsilon CellsUncinate RegionDevelopmental Differences
Citar este artigo
Campbell-Thompson, M. L., Montgomery, E. L., Foss, R. M., Kolheffer, K. M., Phipps, G., Schneider, L., Atkinson, M. A. Collection Protocol for Human Pancreas. J. Vis. Exp. (63), e4039, doi:10.3791/4039 (2012).