新生児マウスの膵臓からの機能性膵島の効果的な分離
Summary
我々は、新生児マウスから無傷の膵島を単離するために、本明細書のプロトコルを記述します。膵臓は、部分的に洗浄し、ハンドピッキング続いて、コラゲナーゼで消化しました。 20から80膵島クラスタは、いくつかの島の研究に適しており、新たに生まれたマウスから膵臓ごとに取得することができます。
Abstract
大型哺乳動物の膵臓からの灌流ベースの膵島分離プロトコルは十分に確立されています。このようなプロトコルは容易に準備ができて、コラゲナーゼ注入とその後の組織潅流を可能にする膵管の大きなサイズのため、多くの研究室で行われています。灌流は小さな膵臓で容易に達成可能ではないので、コントラスト、小さな膵臓から膵島単離では、新生児マウスのような、挑戦的です。ここでは、視覚的な支援を受けて、新たに生まれたマウスからの膵島の相当数を回復する詳細な簡単な手順について説明します。新たに切開全膵臓を微量遠心管中、37℃で、ハンクス平衡塩溶液（HBSS）中に溶解し0.5ミリグラム/ mLのコラゲナーゼIVで消化しました。チューブは、組織の分散を助けるために、定期的にタップされました。組織の大部分を1mmの周りの小さいクラスタに分散させたときに、溶解物を、10％ウシ胎児血清（FBS）で培養培地で3〜4回洗浄しました。アイレットクラスター、認識腺房組織を欠く、その後ステレオスコープを解剖下で回収することができます。新たに生まれたマウスの膵臓あたりの大型小島に80小 – この方法は、20を回収します。これらの島は、インスリン分泌、遺伝子発現、および文化を含むほとんどの考えられる下流のアッセイに適しています。インスリン分泌アッセイの例は、単離プロセスを検証するために提示されています。遺伝的背景と消化の程度が収量を決定する最大の要因です。それは内分泌外分泌分離に役立つように、高い活性を有する新たに作られたコラゲナーゼ溶液が好ましいです。洗浄中のすべてのソリューションやウシ胎児血清中の陽イオンの存在[カルシウム（Ca 2+）とマグネシウム（Mg 2+）] /ピッキングメディアは、適切な整合性を持つ島の良好な収率のために必要です。良好なコントラストおよび倍率の解剖スコープにも役立ちます。
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
ここでは、従来の灌流のためには小さすぎる膵臓から膵島単離にステップバイステップのプロトコルを提供します。このような等 β細胞の精製、遺伝子発現解析、膵島β細胞の成熟、増殖、細胞ストレス応答、細胞生存率、代謝、機能GSISの維持、などのすべての島ベースの研究のための準備ができて膵島をもたらすことが期待されます。これは、我々の知る限りに、新生仔マウスから?…
Offenlegungen
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 |
Referenzen
- 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).
