Name: a murine pancreatic islet cell-based screening for diabetogenic environmental chemicals
Uploaded: 2018-06-25T12:30:00.000+00:00
Duration: 7 min 39 s
Description: Watch this Scientific Journal Video about A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals at JoVE.com

这项工作得到了由 NIEHS 和中国国家自然科学基金 (31572626 号) 赞助的 CREH 中心的试点项目赠款的支持。

所有动物实验都是按照所有相关的指导方针、规章和管理机构执行的。正在示范的《议定书》是在德克萨斯一个 & M 基因组医学研究所的机构动物护理和使用委员会 (IACUC) 的指导和批准下进行的。
1. 解决方案准备
<ol><li>将10x 汉克的平衡盐溶液稀释为 1x, 双蒸馏 H2O, 并贮存在4摄氏度。</li>
	<li>通过添加 HEPES (10 毫米)、氯化镁2 (1 毫米) 和葡萄糖 (5 毫米) 来制备隔离溶液。将 pH 值调整为 7.4, 并保持冰。</li>
	<li>从4型胶原酶中制备胶原酶溶液。 注:具体地说, 胶原酶4型含有 160 U 每毫克的干重。1克胶原酶4型稀释5毫升双蒸馏 H2O 与浓度200毫克/毫升。2毫升的稀释胶原酶添加到30毫升冰冷隔离解决方案, 6 毫升的胶原酶溶液被转移到50毫升管为每只老鼠。</li>
	<li>通过将1毫米 CaCl2添加到隔离溶液中, 准备洗涤液。</li>
	<li>用冷聚蔗糖/钠葡溶液 (1.1119 克/毫升, 5 毫升) 制备纯化溶液。</li>
	<li>将 l-谷氨酰胺 (20 毫米), 青霉素 (100 毫升), 链霉素 (100 µg/毫升) 和胎牛血清 (10%) 加入 RPMI 培养基中, 制备500毫升胰岛培养基。</li>
	<li>在200µM 的蒸馏水中溶解粉末, 并贮存在-20 摄氏度时, 准备 DCFH 的溶液。 注:ROS 染色溶液是通过稀释 DCFH 溶液, 以5µM 的最终浓度在 RPMI 中新鲜地制成的。这种试剂应该保护免受光线的侵害。</li>
</ol>2. 外科准备
<ol><li>腹腔注射 (ip) 一只老鼠与 avertin (2.5% avertin, 0.4 mL/20 g)。当鼠标完全麻醉后, 不再响应后足捏, 弄死鼠标, 移动到生物罩。</li>
	<li>将安乐死鼠标放在腹部面朝上, 用70% 乙醇喷雾皮肤进行杀菌。</li>
	<li>用1厘米的 U 型切口在上腹部打开腹腔, 并将延髓方向的皮肤收回, 露出腹腔。</li>
</ol>3. 胰腺灌注和清除
<ol><li>如图 1所示, 找到壶腹的位置。用一对弯曲钳夹住靠近 Oddi 括约肌位置的十二指肠。用另一对弯曲钳夹住十二指肠壁, 阻断十二指肠的胆汁通路。 注:此步骤中未插入壶腹。这一步骤旨在阻断十二指肠的胆汁通路。</li>
	<li>将鼠标与头部近端和脚远端对研究员进行定位。</li>
	<li>找到胆总管, 用30克针和5毫升注射器慢慢注射3毫升胶原酶溶液。在注射后要确保胆总管和胰腺被充气。</li>
	<li>用弯钳和细剪刀去除膨胀的胰腺, 将其与降结肠、肠、胃和脾分开。将胰腺放置在含有3毫升胶原酶溶液的50毫升管内, 并将其放在冰上。</li>
</ol>4. 胰腺消化
<ol><li>将每50毫升管放入37°c 水浴。孵育 15-20 分钟 (时间随小鼠的应变和年龄而变化)。</li>
	<li>用旋涡震动管子。</li>
	<li>池 4-5 消化胰腺在一个大的无菌水槽过滤器和使用勺子, 使胰腺彻底崩溃。使用23克针和10毫升注射器含有洗涤液, 有力地将消化的细胞从屏幕上移开。在50毫升的管子里收集洗涤液。</li>
	<li>离心管在 97 x g 在4°c 为1分钟和醒酒上清。</li>
	<li>在试管中加入25毫升的洗涤液, 用温和的涡流重新悬浮组织。将组织在 97 x g 处旋转4摄氏度为1分钟, 醒酒上清。重复3次。小心不要把颗粒组织去除。</li>
</ol>5. 胰岛的纯化
<ol><li>将10毫升的纯化液加入洗涤组织颗粒中, 轻轻并用重悬。</li>
	<li>在净化溶液上轻轻覆盖5毫升的隔离液。注意在两个解决方案之间保持一个尖锐的液体接口。</li>
	<li>离心管在 560 x g 在4°c 为15分钟以缓慢的加速度和没有刹车。将吸管插入到界面中, 将胰岛层收集到新的50毫升管中。</li>
	<li>如步骤3.5 所述, 清洗胰岛。</li>
</ol>6. 胰岛细胞的电镀和治疗
<ol><li>并用重悬在10毫升的胰岛培养溶液中的胰岛, 轻轻地混合好, 并放置100µL/井与多通道的吸管到96井板。安置大约5个小岛每96井。</li>
	<li>把盘子放在组织培养孵化器里12小时。</li>
	<li>离心机 (112 x g) 96 井板, 去除培养基。</li>
	<li>在胰岛培养基中稀释环境化学物质。将每种化学稀释剂中的100µL 加入板的分离井中。并用重悬小岛在每个井和孵化12小时在37°c (浓度变化为不同的环境化学物质)。</li>
</ol>7. ROS 染色和测量
<ol><li>胰岛治疗后, 用 1x PBS 冲洗 (每96µL 200 个)。 注:化学物质的浓度如下: AFB1: 3 µM, 阿特拉津: 100 µM, BaP:10 µM, BPA: 100 µM, β雌激素, PCB126:10 µM, 壬基酚和 BPA: 100 µM。</li>
	<li>用 n-乙酰半胱氨酸 (NAC) 作为抗氧化剂来猝灭 ROS。加入5毫米的 NAC 在 ROS 诱导的化学稀释剂和孵育12小时在37摄氏度。</li>
	<li>离心96井板在 112 x g 和孵化与5µM 解答荧光探针 (H2-DCFH-DA 溶解在 RPMI 媒介) 在37°c 为60分钟。</li>
	<li>用 PBS 冲洗处理过的胰岛细胞3次, 用微板块读取器在488纳米上测量荧光。 注:当样品在 488 nm 激发时, 在细胞中的 DCF 的积累可以用荧光量增加 530 nm 来测量。</li>
	<li>执行葡萄糖刺激胰岛素分泌 (GSIS)9的活性氧诱导 xenobiotic 治疗胰岛和未经治疗的胰岛, 以测量 xenobiotic 对胰岛的生理功能的影响。</li>
</ol>

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作者声明他们没有竞争的财政利益。

积累的证据表明, 接触环境化学物质对 T2DM 的发展起着重要的作用。外来化合物诱导的 ROS 已被认为是一个潜在的致病因素, 有助于 T2DM 的发展。人类接触到广泛的 xenobiotic 化学物质, 有很大的需要, 新的研究技术, 以有效地识别胰腺毒物和研究的毒性机制, 特异的胰腺细胞。
在这项研究中, 根据已发布的程序14, 我们已经制定了一个协议, 以隔离鼠胰岛和屏幕的细胞与 xenobiotic 化学物质氧化应激损伤的胰腺细胞。我们还使用这个程序来研究 xenobiotic 化合物引起的胰腺特异性毒性反应。为了从老鼠那里获得胰腺, 我们更喜欢使用 avertin 在其他麻醉, 因为它不会改变血糖水平, 不影响胰腺的血管15,16,17。研究发现, 密度梯度离心法对高纯度胰岛的分离至关重要。应注意获得聚蔗糖/钠和隔离缓冲期之间的不同层, 以获得一个纯胰岛样本, 没有碎片和外分泌细胞/组织。该方法可用于对胰岛生理环境化学效应的体外分析, 如 GSIS 法 (如图 3所示)。胰岛应小心地分散与重复吹打到单独的细胞, 以确保在96细胞培养板上的制服细胞数。这里描述的方法结合了胰岛的分离和快速的活性氧生成法, 因此, 是一个简单有效的初始屏幕, 为潜在的胰腺损伤化学品。
我们使用胰岛素分泌反应葡萄糖挑战, 以确认孤立的胰岛的身份。该程序非常适合分析参数的变化, 包括胰岛素分泌 (图 3) 和阵营生成, 以应对 xenobiotic 治疗18。

T2DM 患病率的上升已成为近年来全球卫生危机, 对公共卫生构成严重威胁1。许多因素被发现与 T2DM 的发展有因果关系, 其中, 反复发现表明, 这些因素的一个共同的收敛点是诱导氧化应激导致过量 ROS 的产生2,3。
广泛的环境化学物质, 包括多氯联苯, 二恶英和 BaP 被发现诱导氧化应激, 这可能会损害胰β细胞的功能, 导致胰岛素抵抗和 T2DM4。虽然 ros 的生理水平在细胞功能中起着重要作用, 但暴露于活性氧超过抗氧化剂系统的能力会导致细胞/组织的损伤, 导致疾病5。胰β细胞表达的抗氧化酶水平较低, 因此是一个敏感的目标为氧化应激介导的损害6,7。慢性暴露于高水平的 ROS 已经表明, 导致应力诱发胰腺细胞功能障碍5以及胰岛素抵抗的肝脏和脂肪组织8。
这个项目的总目标是建立一个细胞为基础的检测, 以筛选化学物质的致糖尿病潜能的基础上, 他们诱导的活性氧在胰腺细胞。胰腺缺乏代谢解毒, 是 xenobiotic 致损伤6、7的敏感靶。因此, 通过直接测定胰腺细胞中产生的 ROS, 可以直接对胰腺内化学诱发的损伤进行近似分析。为了开发这种方法, 我们分离了小鼠胰岛, 用化学物质在细胞培养条件下培养出孤立的胰岛, 并利用化学诱导的 ROS 生成作为读数。该方法简便、有效地鉴别孤立胰岛中的活性氧诱导化学物质;它可以进一步发展, 以调查的毒性机制, 具体的胰腺的体外。

<table><tbody><tr><td>10&amp;times;Hank&amp;rsquo;s balanced salt solution&amp;nbsp;</td><td>GIBCO</td><td>14185-052</td><td></td></tr><tr><td>Collagenase Type 4</td><td>Worthington Biochemical Corporation</td><td>CLS-4</td><td></td></tr><tr><td>polysucrose/sodium diatrizoate solution&amp;nbsp;</td><td>Sigma</td><td>10771</td><td></td></tr><tr><td>2&amp;rsquo;,7&amp;rsquo;-dichlorofluorescein (DCFH-DA)</td><td>Sigma</td><td>D6883-50MG</td><td></td></tr><tr><td>fluorescence microplate reader&amp;nbsp;</td><td>Biotek</td><td></td><td></td></tr><tr><td>L-glutamine</td><td>Sigma</td><td>G8540-25G</td><td></td></tr><tr><td>streptomycin</td><td>GIBCO</td><td>15140148</td><td></td></tr><tr><td>FBS</td><td>GIBCO</td><td>26140079</td><td></td></tr><tr><td>RPMI 1640</td><td>GIBCO</td><td>11875-085</td><td></td></tr><tr><td>avertin</td><td>Sigma</td><td>T48402-25G</td><td></td></tr><tr><td>Rat/Mouse Insulin ELISA Kit</td><td>Millipore</td><td>EZRMI-13K</td><td></td></tr><tr><td>Centrifuge</td><td>Sorval</td><td>Sorval RT7</td><td>for 96-well plate centrifuge</td></tr><tr><td>Microplate reader</td><td>Biotek</td><td>Epoch 2</td><td>for fluorescence reading</td></tr></tbody></table>

a murine pancreatic islet cell-based screening for diabetogenic environmental chemicals

暴露在人类和动物中的某些环境化学物质已经被发现会导致胰腺β细胞的细胞损伤, 这将导致2型糖尿病 (T2DM) 的发展。虽然化学诱导β细胞损伤的机制不清楚, 而且很可能是复杂的, 但一个反复发现是这些化学物质诱导氧化应激导致产生过量的活性氧 (ROS), 从而导致损害β细胞。为了识别潜在的致糖尿病环境化学物质, 我们从 C57BL/6 小鼠和培养的胰岛细胞中分离出了96井细胞培养皿中的胰腺细胞;然后, 用化学试剂对胰岛细胞进行了剂量测定, 用 2 ', 7 '-dichlorofluorescein (DCFH) 荧光染料检测了 ROS 的产生。使用这种方法, 我们发现双酚 a (BPA), 苯并 [a] 芘 (BaP) 和多氯联苯, 可能会诱发高水平的 ROS, 表明它们可能会导致胰岛细胞的损害。该方法应用于筛选致糖尿病外来化合物。此外, 培养的胰岛细胞也可用于胰腺细胞化学诱导毒性的体外分析。

图 2显示了健康的孤立胰岛的显微图像, 其中胰岛具有圆形或椭圆形, 尺寸相对均匀 (虽然大小均匀性可以不同于应变)。我们接下来研究胰岛在试管中的作用, 通过分离胰岛和刺激胰岛素分泌的培养胰岛。图 3显示了我们对由3.3 毫米和16.7 毫米葡萄糖9诱导的 C57BL/6 小鼠孤立胰岛 GSIS 检测的典型分析。
使用板格式屏幕方法, 我们已经发现了几个外来化合物, 导致显着诱导活性氧在胰岛细胞 (图 4)。我们处理了不同化学物质的孤立胰岛12小时, 发现阿特拉津, BPA, BaP 和 PCB126, β雌激素可能导致显着诱导 ROS 在孤立的胰岛10,11,12,13。
虽然 ros 不等于致糖尿病诱导, 有充分的证据表明活性氧对胰腺β细胞的生理功能造成损害的重要作用, 从而导致 T2DM8。因此, 工作的价值是确定这些 ROS 诱导化学作为初始屏幕。此外, 在手稿中描述的孤立胰岛细胞培养系统中, 可以进一步分析所鉴定的化合物。我们已经表明, ROS 诱导外来化合物对葡萄糖刺激胰岛素分泌的孤立胰岛, 简要提供致糖尿病数据, 这些测试化合物。胰岛经 PCB126 和 BPA 预处理后, 胰岛素分泌明显减少 (图 5)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57327/57327fig1v3.jpg"> 图 1: 小鼠胰腺灌注过程.该过程由以下步骤组成: (1) 找到壶腹, 并用两对弯曲钳夹住壶腹。(2) 通过普通胆汁插入针。(3) 在5毫升注射器中慢慢注射3毫升胶原酶溶液。(4) 从十二指肠开始收集扩张的胰腺。 
 
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57327/57327fig2v3.jpg"> 图 2: 孤立的老鼠胰岛.在培养基中显示孤立的小鼠胰岛。刻度条为500µm。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57327/57327fig3v3.jpg"> 图 3: 葡萄糖刺激胰岛素分泌的孤立胰岛.剂量依赖性葡萄糖刺激胰岛素分泌 (1, 3.3, 16.7 毫米, 治疗15分钟) 是测量在孤立的胰岛 (每井平均5个胰岛), 使用鼠标胰岛素 ELISA 试剂盒。误差线显示标准偏差。*p < 0.05。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57327/57327fig4v3.jpg"> 图 4: 从 C57BL/6 小鼠的孤立胰岛中外来化合物诱导 ROS.(A) 对12小时的孤立胰岛进行治疗, 并染色 2 ', 7 '-dichlorofluorescein (DCFH) 1 h 的荧光染料。荧光是由荧光板读取器确定的。(B) 化学诱导的 ROS 由 NAC (5 毫米) 淬火。(AFB1: 3 µM, 阿特拉津: 100 µM, BaP:10 µM, BPA: 100 µM, PCB126:10 µM, 壬基酚, BPA: 100 µM, 和 NAC: 5 毫米)。误差线显示标准偏差。*p < 0.05。<a href="/files/ftp_upload/57327/57327fig4v3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57327/57327fig5v3.jpg"> 图 5: Xenobiotic 对孤立胰岛葡萄糖刺激胰岛素分泌的影响.用 PCB126、β雌激素和 BPA (4 h) 预处理分离胰岛, 并将其保存在胰岛培养基中1小时。用一种商用的小鼠胰岛素 ELISA 法对胰岛上清液进行胰岛素测定。误差线显示标准偏差。*p < 0.05。

Watch this Scientific Journal Video about A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals at JoVE.com

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

在这里, 我们提出了一个协议, 以隔离小鼠胰岛细胞筛选的 ROS 归纳的外来化合物, 以确定潜在的致糖尿病 xenobiotic 化学品。

小鼠胰岛细胞筛选致糖尿病环境化学物质的研究

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

a-murine-pancreatic-islet-cell-based-screening-for-diabetogenic

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.2K 次观看.  Hunan Agriculture University. 在这里, 我们提出了一个协议, 以隔离小鼠胰岛细胞筛选的 ROS 归纳的外来化合物, 以确定潜在的致糖尿病 xenobiotic 化学品。

视频: A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Murine Pancreatic Islet Isolation

Hi, I am Greg Zott. I'm with the UCSF Diabetes Center in the Bluestone Laboratory, and today I'm gonna show you the process for isolating mouse eyelets. The procedure involves the injection of a collagenase solution into the pancreas of a C3 H donor mouse, where we will then remove that pancreas, digest it away, purify the eyelets on a fial gradient, and then handpick them for ultimate transplant into the kidney capsule of a diabetic NOD mouse, Which will be our recipient.Before we start this procedure, we diluted our ATE solution in a modified Hanks buffer and we have adjusted the concentration specific for the strain of mouse that we're using the C3 H.We found digests better at a 0.8 mgs per mil collagenase solution, and we will use that to inject into the common bile duct to distend the pancreas.Performing. The procedure today Will be Pavo codre of the diabetes center. He will inject, open up the mouse, expose the the intestines, and then inject into the common bile duct.We will spray down the mouse with ethanol to keep the hair down, and then we will also verify that the mouse has been sacrificed properly with a toe pinch. Pavo will now open up the mouse and exposing the intestine and the pancreas with a midline incision. The diaphragm will also be cut to verify that the mouse will be sacrificed.Before we get started, I'd like to point out some of the important structures. The stomach is lying right here with the pancreas attached underneath into the duodenum and above. This is the liver where we'll find the gallbladder and the common bile duct, which you can see right here.So what Pavo will do next is he will isolate the small intestine, find where the common bile duct enters the small intestine. Once the intestine is exposed, we clamp on either side of the common bile duct with mosquito clamps. We find that this allows more collagenates to enter the pancreas while other groups try to direct the clamp on top of the common bile duct.We find that this is a better technique in allowing enzyme to enter the pancreas. This is a gentle procedure. You don't want to tear the bowel.You wanna allow the clamps to lie gently on either side of the common bile duct. Now we will find a gallbladder, and once the gallbladder's exposed, we will use that as a guide to injecting the collagenase into the common bile duct. Powell's using a 30 gauge needle with a five cc syringe containing three mills of collagenase solution.As he's entering the common bile duct, he's distending that duct with collagenase solution and then he travels down the common bile duct till he reaches near the intestine. And as he's going down, the enzyme solutions being pumped into the injected into the pancreas. Now that the pancreas is inflated with collagenase solution, Powell's gonna remove the hemostats and then remove the pancreas from the the mouse carefully tearing it away from the intestine.He removes it from the stomach. Okay, and then using the spleen as a handle, he lifts up the pancreas and then slowly snips it away in the common bile duct, and that's it. Pancreas is laid out and then the spleen is removed, and there he's removing the meso enteric tissue, and then the pancreas is placed in a two mill collagenase solution and a sterilized glass vial and it's placed back on Ice.The isolated pancreas that we put into the glass vials will now be placed into a 37 degree water bath for approximately 17 minutes. We placed them into a 50 mil conical rack so that they don't float up. Once during the bath, the timer starts and the digestion begins.Once the digestion time is completed, vials are removed, and then they are shaken and disrupted gently. And once the tissue starts falling apart, the vial is then placed on ice. A sterile screen is placed over a 400 mil beaker that contains washing buffer.The pancreas digest is then carefully poured over the screen into the wash buffer. For demonstration purposes, the hood has been turned off and the glass of the hood has been raised so we could see what we're doing. Once the pancreas digest is poured into the beaker, the vials are rinsed with the same washing buffer and the wash buffer placed over the screen.We have a syringe filled with washing buffer. We will use that to forcefully wash the tissue through the screen. The idea is to release any loose eyelets that are unattached to the matrix, but leaving the membranes and the undigested tissue on the screen.And when you're finished washing a tissue, this is what you usually See at the end of the procedure. The Pancreas tissue that's in the bottom of the tube will now be transferred to two 50 mil conical, and the tissue will be washed, the tissue's mixed evenly, and then distributed evenly so that we have a equal pellet volume in each tube. The beaker is now rinsed with more washing buffer.The sides are rinse so that you get all tissue that is entered the beaker again. Then it's equally distributed between each two, both 50 mil. Conical then are topped off with wash buffer.The tissue is inverted several times and then placed into a centrifuge. This is a quick spin. The centrifuge is allowed to reach a thousand RPM and then is turned off.The tissue at this time is very S eyelets are very sensitive, and you don't want to over compact them. The tubes are removed and we will do this wash step three more times. This is the end of the third wash.The components of the 50 mil conical tube are transferred to the 15 conical tube and centrifuge for a thousand RP M and then stopped. The supernat will be removed and the first part of the FI Call will be started. We are at the step where we Have our pelleted di pancreas digest.We'll resuspend that tissue so it's nice and loose to the pellets. We'll add the first density gradient, which is 1 1 0 8. We'll add five mils to each tube.The pellet is then mixed thoroughly in each of the gradients. It's important that you get good tissue distribution in your heaviest gradient. Now we will layer two mils of 1 0 9 6 density.We will then on top of that, layer two mils of 1 0 6 9 density, and then on top of that 1 0 3 7 density. It's important while you're layering the densities that you don't mix. Then all the layers have been added to the tube.And you can see this is the top of 1 1 0 8, the top of 9 1 0 9 6, the top of 1 0 6 9 and the top of 1 0 3 7. And you could see the tissue is already starting to move to its proper density even without centri fusion. The fial gradients are brought up to speed at 1800 RPM for 15 minutes, and remember to keep the brake off after the tubes are removed from the centrifuge.You can see between the 0 0 9 6 and 0 0 7 layer, there's a nice band of eyelet tissue Pablo's. Now removing the 0 3 7 layer to remove any tissue debris and DNA that might have been released during the processing. Using a sterile plastic pasture pipette, he will now remove the islet layer and transfer them to a 50 mil conical containing 25 mils of wash buffer.You want do this step quickly because fol is toxic to eyelets. We actually removed the entire 0 6 9 and 0 9 6 layer. Pavo cuts the top of the plastic pasture pipette and rinses it with wash buffer.The eyelets are then centrifuge like we centrifuge previously. Centrifuge is brought up to a Thousand RPM and then we stop it and We aspirate off. The supernatant eyelets will be washed three times with wash media.So we just completed the eyelid Isolation process we took from the digest to the fol gradient to the purified eyelets after our third wash with wash buffer, we resuspended the eyelets in our RPMI media and we played it in a tissue culture plate. These eyelets are now ready for in vitro assays or in vivo preps like eyelet Transplant.

小鼠胰岛隔离

科研

生物学

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

支架支持胰岛移植糖尿病小鼠附睾脂肪垫

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

生物工程

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

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.

瘦素受体<sup&gt;分贝</sup&gt; 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

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

医学

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

胰岛门静脉移植小鼠模型的研究

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

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 ...

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared from live human and mouse pancreatic tissue embedded in agarose and cut using a vibratome. This method allows for the tissue to maintain viability and function in addition to preserving underlying pathologies such as type 1 (T1D) and type 2 diabetes (T2D). The slice method enables new directions in the study of the pancreas through the maintenance of the complex structures and various intercellular interactions that comprise the endocrine and exocrine tissues of the pancreas. This protocol demonstrates how to perform staining and time-lapse microscopy of live endogenous immune cells within pancreatic slices along with assessments of islet physiology. Further, this approach can be refined to discern immune cell populations specific for islet cell antigens using major histocompatibility complex-multimer reagents.

This study presents the application of live pancreatic tissue slices to the study of islet physiology and islet-immune cell interactions.

观察活胰腺组织切片中的胰岛功能和胰岛-免疫细胞相互作用

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared ...

免疫与感染

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

利用邻近结扎测定法在人类胰腺β细胞中原位内源性米托巴结物的可视化

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

In this protocol, a method of murine islet isolation and transplantation into the inguinal subcutaneous white adipose tissue is described. Isolated syngeneic murine islets are transplanted into a murine recipient using a basement membrane hydrogel. The blood glucose level of the recipients is monitored, and histology analysis of the islet grafts is performed.

毛里岛皮下白脂肪组织（ISWAT）移植模型

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

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 ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

小鼠胰腺胰岛细胞亚群中的钙动力学成像

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...