新生小鼠胰腺胰岛功能的有效隔离
Summary
我们在此描述了一个协议，从新生小鼠分离胰岛完好无损。胰腺被部分用胶原酶消化，接着洗涤和手采摘。 20 – 80胰岛集群可以每胰腺从新生小鼠，这是适合于几个胰岛研究而获得。
Abstract
从大型哺乳动物的胰腺基于灌注胰岛隔离协议确立。这种协议是很容易地在许多实验室由于大尺寸的胰管，允许准备注射胶原酶和随后的组织灌注的进行。与此相反，从小型胰腺胰岛分离，像新生小鼠，是具有挑战性，因为灌注是不容易在小胰腺实现的。在这里，我们描述了从恢复视觉协助新生小鼠胰岛相当数量的详细的简单过程。新鲜解剖整个胰腺用在37℃下溶解在Hanks'平衡盐溶液（HBSS）0.5毫克/毫升胶原酶IV消化，在微量离心管。管定期窃听，以帮助组织扩散。当大部分组织的分散，以小簇约1毫米，裂解物，用10％胎牛血清（FBS）洗涤三到四倍用培养基。胰岛集群，缺乏可识别的腺泡组织，然后可以根据解剖立体镜来回收。这种方法恢复20 – 80小到每个刚出生的小鼠胰腺大型小岛。这些胰岛适合大多数可想到下游测定法，包括胰岛素分泌，基因表达，和培养。呈现胰岛素分泌测定法的实例，以验证隔离过程。遗传背景和消化的程度是决定产量的最大因素。高活性新鲜的胶原酶溶液是首选，因为它在内分泌外分泌隔离艾滋病。阳离子在洗涤所有的解决方案和胎牛血清存在下[钙（Ca 2+）和镁（Mg 2+）] /拾取介质所必需的具有适当的完整性胰岛的良好的产率。具有良好的对比度和放大倍率解剖范围也将有所帮助。
Introduction
Isolating pure pancreatic islets is essential for assaying glucose stimulated insulin secretion (GSIS) of beta cells and for islet transplantation from cadaveric donors 1-3. It is also necessary to establish endocrine gene expression in islet cells 4, 5. For this purpose, detailed protocols have been established to allow for isolation of pancreatic islets from large pancreata (6 and references therein). These methods are based on enzymatic perfusion to dissociate acinar from islet tissues, coupled with gradient separation and hand picking. Thus, islet isolation from large pancreas can be performed readily in most laboratories. On the other hand, no detailed step-by-step protocol exists to allow for the isolation of islets from pancreata that are too small to perfuse.
Studying gene expression and function of neonatal islets is important. Neonate islets have different properties from adults in insulin secretion and proliferation capability 7, 8. However, isolating islets from newly born animals, especially mice is challenging due to the small size of the newly born pancreas. The size prevents the usual perfusion process when collagenase is injected though the pancreatic duct. Indeed, several papers have presented studies along these lines, with enzyme or non-enzyme aided isolation procedures 7, 9, 10. However, detailed description of the islet isolation process with visual aid is lacking 7, 9, making it a challenge for most researchers to perform similar studies.
We have explored several different conditions that yield high quality islets from neonatal mice. Here we present a protocol that is expected to help researchers learn the key details in the islet isolation process. This protocol is applicable to mouse pancreas up to two weeks of age, after which perfusion can be performed for routine islet isolation. Islets can be directly used for insulin secretion and gene expression assays.
Protocol
Representative Results
Discussion
在这里，我们提供胰岛分离的一步一步的协议，从是传统灌注太小胰腺。据预计产量胰岛准备好所有基于胰岛研究，如β细胞的纯化，基因表达分析，胰岛β细胞的成熟，增殖，细胞应激反应，细胞存活，代谢和功能GSIS维护等 。这将是，以我们所知，第一次详细目视协议，引导新的研究人员从新生小鼠进行胰岛分离的。如果没有这些视觉助手，广泛的试验和错误是预期的大多数研究人员实?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by grant from NIDDK (DK065949 for GG). We thank Dr. Brenda M. Jarvis and Jeff Duryea Jr. for reading the manuscript.
Materials
| Collagenase Type IV | Sigma Aldrich, St Louis, MO | C5138 | |
| 1X HBSS with Ca2+/Mg2+ | Mediatech/Cellgro, Manassas, VA | MT21020CV | If HBSS wihout Ca2+/Mg2+ is obtained, CaCl2 and MgSO4 can be added to 1.26 and 0.5 mM, respectively. |
| RPMI 1640 w/o glucose | Thermo Fisher Scientific/Life Technologies, Waltham, MA | 11879-020 | Glucose needs to be added to specific levels to not interfere with seusequent islet usage. |
| Glucose | Sigma Aldrich, St Louis, MO | G-7021 | |
| polysucrose and sodium diatrizoate solution | Sigma Aldrich, St Louis, MO | Histopaque-10771 | |
| Stereoscope | Carl-Zeiss, Oberkochen, Germany | Stemi2000 | |
| Stereoscope | Leica, Wetzlar, Germany | Leica M165 | |
| Microcentrifuge | Eppendorf, Hauppauge, NY | Centrifuge 5417C | |
| Centrfuge | Eppendorf, Hauppauge, NY | Centrifuge 5810R | |
| 15-ml centrfuge tubes | VWR, Radnor, PA | 89039-666 | |
| 50-ml centrfuge tubes | VWR, Radnor, PA | 89039-658 | |
| Precision balance | VWR, Radnor, PA | VWR-225AC | |
| Microfuge tubes | VWR, Radnor, PA | 87003-294 | |
| Pipetman P1000 | Fisher Scientific, Waltham, MA | F123602 | |
| Pipetman P20 | Fisher Scientific, Waltham, MA | F123600 | |
| 100X15 millimeter dish | VWR, Radnor, PA | 25384-088 | |
| 60X15 millimeter dish | VWR, Radnor, PA | 25384-168 | |
| 12-well plates | VWR, Radnor, PA | 665-180 | |
| Scissor | Fine Scientific Tools, Foster City, CA | 14080-11 | |
| Tweezers | Fine Scientific Tools, Foster City, CA | 5708-5 | |
| CD1 mice | Charles River Laboratories, Wilminton, MA | CD-1 | |
| C57BL/6J | The Jackson laboratory, Farmington, CT | C57BL/6J | |
| B6CBAF1/J | The Jackson laboratory, Farmington, CT | B6CBAF1/J |
References
- Matsumoto, S., et al. Improvement of pancreatic islet cell isolation for transplantation. Proc (Bayl Univ Med Cent). 20 (4), 357-362 (2007).
- Zmuda, E. J., Powell, C. A., Hai, T. A method for murine islet isolation and subcapsular kidney transplantation. J Vis Exp. (50), (2011).
- Zhao, A., et al. Galphao represses insulin secretion by reducing vesicular docking in pancreatic beta-cells. Diabetes. 59 (10), 2522-2529 (2010).
- Planas, R., et al. Gene expression profiles for the human pancreas and purified islets in type 1 diabetes: new findings at clinical onset and in long-standing diabetes. Clin Exp Immunol. 159 (1), 23-44 (2010).
- Tonne, J. M., et al. Global gene expression profiling of pancreatic islets in mice during streptozotocin-induced beta-cell damage and pancreatic Glp-1 gene therapy. Dis Model Mech. 6 (5), 1236-1245 (2013).
- London, N. J., Swift, S. M., Clayton, H. A. Isolation, culture and functional evaluation of islets of Langerhans. Diabetes Metab. 24 (3), 200-207 (1998).
- Rorsman, P., et al. Failure of glucose to elicit a normal secretory response in fetal pancreatic beta cells results from glucose insensitivity of the ATP-regulated K+ channels. Proc Natl Acad Sci U S A. 86 (12), 4505-4509 (1989).
- Hellerstrom, C., Swenne, I. Functional maturation and proliferation of fetal pancreatic beta-cells. Diabetes. 40 (Suppl 2), 89-93 (1991).
- Blum, B., et al. Functional beta-cell maturation is marked by an increased glucose threshold and by expression of urocortin 3. Nat Biotechnol. 30 (3), 261-264 (2012).
- Hegre, O. D., et al. Nonenzymic in vitro isolation of perinatal islets of Langerhans. In Vitro. 19 (8), 611-620 (1983).
- Dolenšek, J., Rupnik, M. A., Stožer, A. Structural similarities and differences between the human and the mouse pancreas. Islets. 7 (1), e102445 (2015).
- White, J., White, D. C. . Source Book of Enzymes, Source Book of Enzymes. , (1997).
- Bock, T., Pakkenberg, B., Buschard, K. Genetic background determines the size and structure of the endocrine pancreas. Diabetes. 54 (1), 133-137 (2005).
- Bouwens, L., Rooman, I. Regulation of pancreatic beta-cell mass. Physiol Rev. 85 (4), 1255-1270 (2005).
