Name: a method for mouse pancreatic islet isolation and intracellular camp determination
Uploaded: 2014-06-25T13:00:00
Description: Watch this Scientific Journal Video about A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination at JoVE.com

我们要感谢蕾妮L. Pasker和HARPREET K.布拉尔就在这项工作中所描述的协议，专家技术援助。此外，我们要感谢克里斯托弗B. Newgard的师徒在杜克大学和Alan D. ATTIE在威斯康星大学麦迪逊分校，连同他们的实验室的成员，这使得我们的时间的支持，并支持必要的优化描述协议。具体而言，我们感谢汉斯Hohmeier，丹红路和海伦娜温菲尔德在Newgard实验室和玛丽Rabaglia在ATTIE实验室富有成效的讨论和建议。这项工作是由美国国立卫生研究院授予DK080845和青少年糖尿病594支持 研究基金会赠款17-2011-608（以MEK）

所有动物实验均符合所有相关指引，法规和监管机构执行。威斯康星 - 麦迪逊大学的机构动物护理和使用委员会（IACUC）的指导和批准下进行演示的协议。 1，准备解决方案<ol><li>安乐死在本协议的方法是放血下圣阿韦坦麻醉（替代品的审查，见讨论）。使圣阿韦坦，在15毫升锥形管和热加0.625克2-2-2三溴乙醇的（1.25％或44.2毫米）到1.25毫升2 - 甲基-2 - 丁醇，在37℃下进行20-30...

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糖尿病患者预计将影响到世界人口的7.7％的患病率，新颖的研究方法的要求是必须既认识和对待糖尿病18。目前胰岛分离是用于体外实验完善的协议，并已稍作修改11,14,16以前提出。虽然胰岛素分泌的胰岛共同的下游应用，专注于上游的成分，如环磷酸腺苷，可以帮助划定增效胰岛素的产生和分泌的机制。 安乐死的选择必须作出深思熟虑，以确保双方的...

严格的血糖正常的维护是必要的，以防止并发症如神经病，肾病，视网膜病变和，这是不受控制的1型和2型糖尿病1的病理学的所有特点。减少β细胞功能和质量的1型和2型糖尿病扰乱血糖浓度2。而从产生胰岛素的β细胞，受损的β-细胞分泌胰岛素，并在型外围胰岛素信号2型糖尿病的毁灭性损失免疫介导的1型糖尿病患者的结果一起促进高血糖，血脂异常，并增加肝葡萄糖生成，最终导致既损失的β-细胞质量和胰岛素分泌能力从个体β-细胞3。理解在1型和2型糖尿病的进展相关的β-细胞的机制可望产生新的治疗，预防和治疗这些疾病。 体外 TISSUE培养模型，如INS-1和MIN6永生化的β-细胞系，可用于理解特定的β-细胞功能的有用工具。然而，在胰岛内的不同细胞类型的相互作用本身可调节β-细胞的功能。例如，胰高血糖素的旁分泌的影响（解除α-细胞）和生长抑素（从δ-细胞释放）的增加和减少胰岛素的分泌，分别展示了在内分泌反应4芯电池接近的重要性。此外，β-细胞之间的间隙连接增强胰岛素5的释放。此外，虽然进步已在产生胰岛素线路，更好复制的分离胰岛的生理响应葡萄糖（ 例如 ，INS-1衍生十三 ​​分之八百三十二和3分之832细胞系），它们的葡萄糖响应性仍不同于正常大鼠胰岛6,7。此外，这些克隆胰岛素瘤细胞系的反应以胰高血糖素样肽-1（GLP-1）受体激动剂可以从彼此显着不同，以及从正常胰岛6。因此，永生化细胞系可能并不代表用于测定药物对cAMP生成的影响的最佳模式。 与此相反的胰岛来源的细胞系，仅在整体动物模型研究胰岛β细胞功能提供了它自己的一套并发症。其中与内分泌组织工作面临的最大挑战是测量激素的精确浓度释放。具体地说，肝起着重要的作用代谢胰岛素和胰腺的血流直接进入肝脏。因此，血浆胰岛素的测量可能不准确地描绘胰岛素从胰腺自身或不同处理对胰岛素分泌8的速率的影响被分泌的量。另外，胰高血糖素的肾代谢可能限制胰高血糖素输出的可靠度由胰岛α-细胞9。因此，初级分离小鼠胰岛体外实验提供了如何胰岛是响应特定刺激，以补充体内的测量结果更准确的理解。 本协议的小鼠胰岛的分离是通过使用一组数（有轻微的修改，可能有助于提高成功）10,11行之有效的协议。另外，cAMP产生的确定允许直接读出的β-细胞的肠降血糖素应答性。与cAMP的测定，蛋白质含量和胰岛素分泌相结合，也可以从相同的cAMP的样品制备量化，帮助确定在β细胞功能的缺陷是否位于近端或远端的cAMP 10。最后cAMP含量及在本协议中胰岛素分泌的应用程序可以是一个非常强大的工具，为了解医药和饮食成分的影响，其中包括秒，对cAMP和胰岛素分泌。除了 ​​刺激从单独的葡萄糖，其它的化合物可用于测量变化的cAMP和胰岛素分泌10,11。 最后，虽然胰岛素是我们从分离胰岛，其他激素，如胰高血糖素和促生长素抑制素，以及细胞因子，类二十烷酸，和环磷酸腺苷测定的主要激素，也可以测量，无论是由瞬态刺激测定法或通过定量它们的含量在培养基12。最后，虽然本手稿的范围之外，胰岛分离与所述胶原酶分离方法允许对胰岛保存，使得许多其它下游应用可以追求，如胰岛移植，RNA提取用于定量实时PCR或微阵列分析，蛋白隔离蛋白印迹，胰岛嵌入和免疫荧光成像，和[3H] - 胸苷掺入如胰岛细胞REPLI的量度阳离子，其中一些在前面的朱庇特的文章13-16进行了描述。总体而言，继在协议中所述的胰岛分离过程中可提供研究者与开发的疗法，促进药物发现旨在提高胰岛β细胞功能的重要和有用的信息。

<table border="0" cellpadding="0" cellspacing="0" width="502">
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	<tbody>
		<tr height="64">
			<td height="64" style="height:64px;width:235px;">Collagenase: Collagenase from Clostridium histolyticum suitable for isolating active islets</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">C7657</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Ficoll 400</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>F9378</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Hanks Balanced Salt Solution 10X</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td>14065-056</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Hepes</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>H3375</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">RPMI 1640 (powder)</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td style="width:136px;">31800-022</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Albumin from Bovine Serum (BSA)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>A7888</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">3/0 Silk Suture Thread</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td>18020-30</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Dumont #5 Forceps</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td>11251-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">0.8 mm Forceps&#160;&#160;</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">11050-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Curved Scissors</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">14061-10</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:235px;">Vannas-T&#252;bingen Spring Scissors - Straight/Sharp/8.5 cm/5 mm Cutting Edge</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:136px;">15003-08</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Dissecting Scissors</td>
			<td style="width:131px;">Fine Science Tools&#160;&#160;</td>
			<td style="width:136px;">14002-14</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">5ml BD Luer-Lok Syringe</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">309646</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">1ml BD syringe</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">309628</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">30 G BD Needle 1/2&#34; Length</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">305106</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">27 G BD Needle 1/2&#34; Length</td>
			<td style="width:131px;">BD</td>
			<td style="width:136px;">305109</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Sharpening Stone</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:136px;">29008-01</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">2-2-2 tribromoethanol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">T48402-25G</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">2-methyl-2-butanol</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>240486-100mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Sodium Chloride (NaCl)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>S9888</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Potassium Chloride</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td>P3911</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:235px;">Monopotassium Phosphate (KH2PO4)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">P0662</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">Sodium Bicarbonate (NaCHO3)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">S6014</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">CaCl2 *2H2O</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">C3881</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:235px;">MgSO4 *7H2O</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">M9397</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">Penicillin-Streptomycin</td>
			<td style="width:131px;">Invitrogen (Gibco)</td>
			<td style="width:136px;">15140-122</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:235px;">Heat Inactivated Fetal Bovine Serum (H.I. FBS)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:136px;">SH30088.03HI</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:235px;">3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:136px;">5879-100MG</td>
		</tr>
	</tbody>
</table>

a method for mouse pancreatic islet isolation and intracellular camp determination

不受控制的高血糖是糖尿病的标志，并促进像神经病，肾病，视网膜病和发病率。患有糖尿病，这两种免疫介导1型和肥胖相关的2型，研究目的是区分糖尿病的病理生理和治疗机制的日益普及是至关重要的。 β-细胞朗格汉斯的胰岛负责适当地分泌胰岛素响应于升高的血糖浓度。除了葡萄糖等营养物质，β-细胞被特定的激素也刺激，称为肠促胰岛素，这是从肠道响应对β-细胞受体增产细胞内环磷酸腺苷一顿饭和行为分泌（ cAMP）的。减少β细胞的功能，质量和肠促胰岛素反应是很好理解有助于2型糖尿病的病理生理机制，并且也正在越来越紧密无线第1型糖尿病。本小鼠胰岛分离和cAMP测定的协议可以是一个工具，以帮助界定促进疾病进展和治疗干预，特别是那些由肠降血糖素受体介导的或相关的，通过细胞内cAMP产生的调制作用的受体机制。而仅cAMP的测量将要描述的，所描述的胰岛分离协议创建一个清洁制剂，其还允许在许多其他的下游应用，包括葡萄糖刺激的胰岛素分泌，[3 H] -胸苷掺入，蛋白质丰度，和mRNA的表达。

为了确保隔离在一个高胰岛产量，在协议中所概述的手术技术应密切随访。虽然这里介绍的技术将针对每个实验室有，这将导致一个成功隔离了几个关键步骤。为了使胆总管方便，所以建议的器官被移位到右侧的鼠标（ 图1）。此外，这将使胰腺膨胀以较小的阻力，因为会有较少的重量限制了业务发展。另一个关键步骤，以最大限度地提高胰岛产量胆总管接近奥狄括约肌（ 图2A-...

Watch this Scientific Journal Video about A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination at JoVE.com

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

含量测定采用胰岛分离的小鼠胰岛体外 β细胞功能是在糖尿病病理生理学和治疗学研究的一个重要组成部分。虽然许多下游应用的情况下，该协议特别描述作为必要参数确定的β-细胞功能的细胞内环磷酸腺苷（cAMP）的测定。

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Research

JoVE Journal

Biology

Murine Pancreatic Islet Isolation

小鼠胰岛隔离

科研

生物学

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

人类胰岛的分离：第一部分：收集和胰腺组织消化

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

人类胰岛的分离：第二部分：人类胰岛的分离纯化及文化

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

计算机辅助大型胰岛质量，粒度分布和结构的可视化和量化

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

小鼠胰岛的朗格汉斯的分离纯化的胶原酶和中性蛋白酶结合使用

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

近红外（NIR）光增加线粒体功能的标志物的表达，在鼠标前庭上皮细胞

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

胰腺胰岛与小鼠分离的简单高效协议

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

A Method for Islet Transplantation to the Omentum in Mouse

小鼠骨髓胰岛移植的一种方法

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet ...