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?...

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。
<p ...

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

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

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

小鼠胰腺内分泌细胞的单细胞 Transcriptomic 分析

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

发育生物学

Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate the measurement of islet oxygen consumption using a 96-well microplate designed for the measurement of oxygen consumption in spheroids. In this assay, spheroid microplates are coated with a cell and tissue adhesive on the day prior to the assay. We utilize a small volume of adhesive solution to encourage islet adherence to only the bottom of the well. On the day of the assay, 15 islets are loaded directly into the base of each well using a technique that ensures optimal positioning of islets and accurate measurement of oxygen consumption. Various aspects of mitochondrial respiration are probed pharmacologically in non-human primate islets, including ATP-dependent respiration, maximal respiration, and proton leak. This method allows for consistent, reproducible results using only a small number of islets per well. It can theoretically be applied to any cultured spheroids of similar size.

This protocol demonstrates the accurate and reproducible measurement of oxygen consumption in non-human primate pancreatic islets. The islet loading techniques and coating of the microplate provide a framework for efficient measurement of respiration in other types of cultured spheroids.

非人类灵长类动物胰腺胰岛耗氧量分析

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate ...

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

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

高含量<em&gt;体外</em&gt;胰岛β细胞复制发现平台

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

用于测试胰岛素模拟化合物的蛋模型

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

神经科学

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 ...

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 ...

小鼠胰腺 β 细胞中线粒体自噬的定量分析

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in &#946;-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify &#946;-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for &#946;-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in &#946;-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous &#946;-cell models.

作者聚焦:使用有效且稳健的互补方法评估胰腺 β 细胞中的线粒体自噬通量 

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic &#946;-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

研究胰腺β细胞线粒体自噬通量的互补方法

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient ...