瘦素受体<sup>分贝</sup> 2型糖尿病小鼠模型：胰岛分离和活细胞双光子成像完好胰岛
Summary
We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.
Abstract
2型糖尿病是一种慢性疾病，影响到3.82亿人，2013年，预计将上升到5.92亿，到2035年1。在过去的二十年中，β细胞功能障碍的2型糖尿病患者已明确规定2的作用。研究进展所需的胰岛的分离方法。胰岛分离的协议这里提出股与来自其它组协议许多共同的步骤，具有一些修改，以提高来自野生型和糖尿病瘦素受体分贝 （的db / db）小鼠分离的胰岛的产量和质量。的实时小区2光子成像方法被提出可用于研究胰岛素分泌胰岛内的控制。
Introduction
在疾病的β细胞功能障碍中的作用已被广泛认可3,4。细胞系如MIN6和INS-1是有用的工具来理解的β细胞行为的生物学。然而，在生理控制胰岛素分泌的需要胰岛内进行。这些胰岛包含数千紧密包装的β细胞，以及血管和其它内分泌细胞类型。胰岛内这样的环境影响胰岛素分泌和很可能是在糖尿病重要。因此，要理解的生理控制胰岛素分泌，以及疾病的病理生理学，有必要研究完整的胰岛。
胰岛分离
从2型糖尿病患者活人胰岛和，特别是人胰岛是很难获得的。此外，人类的胰岛有实验分子操作的可能性有限。因此，研究人员使用的是允许从动物和2型糖尿病的动物模型。一个这样的疾病模型是的db / db小鼠。这是模型2型糖尿病的表型发展的非常类似于人类疾病5,6自发突变。从糖尿病db / db小鼠呈现此处胰岛分离的协议具有许多共同的步骤与其它基团的一些精炼为更好产量，纯化和增强胰岛存活。
2光子成像
此处描述的活细胞2光子检测使研究人员能够量化数和从糖尿病7和野生型胰岛8,9-许多细胞评估单含胰岛素的颗粒的特征。
Protocol
Representative Results
Discussion
在胰岛分离中最关键的因素是胰腺的初始灌注;一个下灌注胰脏结果在相当低的胰岛的收获量。其它因素也影响了隔离质量如消化时间和摇晃可能部分弥补灌注的水平的水平。例如，一个完全灌注胰脏将需要在37℃下被孵育〜18分30秒，轻轻振动，与此相反的下消化胰，可能需要约20分钟的消化与较硬晃动。两种酶消化我们手中的混合物提供更好的胰岛分离比任何一个单独的。对于胶原酶唯一方法，?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by an Australian Research Council Grant DP110100642 (to PT) and National Health and Medical Research Council Grants APP1002520 and APP1059426 (to PT and HYG).
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Liberase TL 5mg | Roche | -5401020001 | |
| Collagenase type IV-1g | Gibco-Life Technologies | 17104-019 | |
| Histopaque 1077 500ml | Sigma Aldrich | 10771 | |
| RPMI 1640 Medium (10X) 1L | Sigma Aldrich | R1383 | to prepare isolation media |
| RPMI 1640 (1X) 500ml | Gibco-Life Technologies | 21870-076 | to prepare cultured media |
| Penicillin-Streptomycin 100ml | Gibco-Life Technologies | 15140-122 | to prepare cultured media |
| Fetal bovine serum 500ml | Gibco-Life Technologies | 10099-141 | to prepare cultured media |
| DMEM (Dulbecco's Modified Eagle Medium) | Gibco-Life Technologies | 11966-025 | to dilute the liberase |
| Metamorph program | Molecular Devices, USA | to analyze the 2-photon images |
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