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).

注:所有本实验是按照昆士兰大学的地方动物伦理规程（经昆士兰大学生物科学解剖伦理委员会）执行。 1.胰岛分离<ol><li>试剂的准备<ol><li>酶的混合物<ol><li>对于胰腺消化，使用LIBERASE和IV型胶原酶的混合物。稀释LIBERASE TL（嗜热低）1瓶5毫克用26毫升的DMEM（贝科改良Eagle培养基）。 </li><li>准备在Hank氏缓冲IV型胶原酶在浓度为0.5mg / ml的，补充有5mM的HEPES，0.5mM的氯化钙 ，为0.1mg / ml的DNA酶和1mg / ml的牛血清白蛋白。 </li><li>混合LIBERASE TL和胶原酶溶液以4:1的比例（按体积计）， 即加入4ml LIBERASE TL +1毫升胶原酶。等分的酶的混合物在-20℃至2.5毫升每个和存储。 注:该组织孵化时间vARY酶批次之间的位（30秒到1分钟差）。因此，一批测试可能需要。 </li></ol></li><li> RPMI媒体隔离<ol><li>溶解的RPMI 1640中粉一个小瓶在1升水中在RT和补充与2g 碳酸氢钠 ，4.02克HEPES。 </li><li>结合的RPMI隔离介质在一个大锥形瓶中，用磁力搅拌器混合，调节至pH7.4，用NaOH，过滤灭菌，并在4℃下存储。 </li></ol></li><li> RPMI文化传媒</li><li>使用的RPMI培养基，含10％胎牛血清和1％抗生素（ 即 ，青霉素100U /毫升，链霉素100微克/毫升）。 </li></ol></li><li>胰岛分离和培养<ol><li>牲<ol><li>牺牲小鼠根据当地机构的伦理程序颈椎脱位或CO 2窒息。用70％乙醇彻底喷鼠标主体。 </li></ol></li><li>胰腺灌注<ol><li> ü本身2长切口横向穿过腹部皮肤和腹膜，使V形翼板。拉扯皮肤向上的襟翼揭开整个腹腔，抛开肠和保持就位肝经折叠薄纸以显示胆总管（ 图1和2）。把动物使头部朝向外科医生和尾部远离外科医生。 </li><li>应用血管钳在壶腹在十二指肠壁进入十二指肠（ 图1）阻断注射酶。 注意:这是一个重要的步骤，因为它决定了注入酶是否将进入胰管和灌注胰脏或回流到注射部位（超过钳位）或进入十二指肠（下夹子）。 </li><li>导管插入胆总管有一个31 G针肝总管和胆囊管（ 图1）的交界处附近，留有余地，第二次注射，如果日Ë胰腺不能灌注的第一次。慢（超过约10秒）注​​资约冷酶混合物2毫升到小鼠胆总管。 注:胰腺将很快灌注如果夹具是在正确的位置。 </li><li>调整夹子的位置，如果所述针是胆总管内但不能灌注胰脏（ 即 ，从肝脏如果超过夹紧向上移动夹具有点远或移动下来一点到肝脏如果下夹紧）。 注:针有时进入周围胶囊而非导管的内腔中。一种有用的标志来检查针是否是内部胆总管是导管注射时的膨胀。如果胰脏不能灌注，另一种是酶混合物可以注入到具有较低产量预期胰腺的多个站点。胰腺（脾叶）的左侧部位的灌注是胰岛的收获量的最大化很重要的。 </li><li>后胰腺为perfused，迅速取出胰腺，放入小瓶（约20毫升，玻璃）和孵育〜19分钟，在37℃水浴。 注:孵育时间可以改变一点取决于小鼠品系和年龄。 </li><li>后的培养时间，加入约20毫升冷的隔离介质的停止消化过程。摇动小瓶轻轻地破坏胰腺，并通过正常的茶筛（1毫米孔径）倒入一个无菌的50ml管中。顶部与隔离媒体和离心管400 XG在4℃下1分钟，摘下。 </li><li>再次溶解在隔离介质颗粒与传输两个15ml试管，然后离心，在400×g离心1分钟，在4℃下与制动切断。吸出上清液后，通过涡旋或上下抽吸混合两种粒料以及用6ml密度梯度细胞分离介质（的Histopaque 1077溶液）每管中。然后轻轻加〜6毫升隔离介质在管的侧面上的顶密度梯度分离介质和离心机在100×g离心15分钟，在4℃下与制动切断。 </li><li>收集在上部（分离介质）上清，中部和下部（密度梯度细胞分离介质）层在3个不同的菜肴。精选在这些三个培养皿胰岛，用解剖显微镜下的吸移管。 注:中间层包含了大多数的胰岛。胰岛被认为是约50至500微米的尺寸紧凑的结构（ 图3）。我们获得〜200胰岛在WT小鼠和db / db小鼠300- &lt;100小岛。 </li></ol></li><li>胰岛培养<ol><li>为了帮助胰岛从酶消化回收，40胰岛中的25cm培养皿6毫升的RPMI培养基中（10mM葡萄糖）培养基，在37℃，5％/ 95％的CO 2 / O 2]为前活2天-cell双光子成像。每天更换培养基。 </li></ol></li></ol></li></ol> 2。活细胞-2-光子成像<ol><li>试剂准备<ol><li>外缓冲区<ol><li>制备的钠丰富细胞外缓冲器用于与140 mM氯化钠，5mM的氯化钾，2.5mM的氯化钙 ，1mM的MgCl 2的，5mM的碳酸氢钠和10mM HEPES的2光子检测。 </li></ol></li><li>外染料<ol><li>对于双光子测定中，准备股票SRB 8mM的胞外缓冲液，分装成小管并储存在-20℃。稀释股票SRB 1:10新鲜外缓冲器得到的0.8毫米的工作浓度。 </li></ol></li></ol></li><li>光子成像<ol><li>首先，预孵育2天的培养的胰岛在3毫米的葡萄糖胞缓冲液30分钟。 注:我们在培养2至5天使用的小岛。 </li><li>然后，将载玻片上的热控制腔装在显微镜载物台上。使用润滑脂以防止溶液泄漏。 </li><li>覆盖用500μl细胞外的腔室ular缓冲器含有0.8毫SRB具有不同葡萄糖浓度（3mM的，6毫15 20mM和20mM葡萄糖）。 </li><li>下一步，将预孵育的胰岛到室的中部和聚焦在3-4细胞层进入胰岛，其中，在啮齿动物胰岛，免疫染色胰岛素表明大部分细胞是β细胞。 注:SRB将概述胰岛所有细胞膜迅速，胰岛准备图像。 </li><li>使用双光子显微镜用60X油浸物镜（NA 1.42）和图像处理软件（ 例如 ，ScanImage 11）。 图4示出了一个典型的显微结构。在550-650纳米的检测采用SRB作为膜透性荧光标记物外，在880 nm光激发飞秒激光脉冲图片胞吐事件，有荧光。记录胰岛反应的刺激。 </li><li>由小荧光点的突然出现识别单胞吐事件（分辨率10像素/1;米），并通过荧光信号的快速上升阶段确认在感兴趣的区域）（〜0.78μm2以下。 </li></ol></li></ol>

<ol>
	<li>Guariguata, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+estimates+of+diabetes+prevalence+for+2013+and+projections+for+2035.">Global estimates of diabetes prevalence for 2013 and projections for 2035.</a> Diabetes Research and Clinical Practice. 103, 137-149 (2014).</li><li>Ashcroft, F. M., Rorsman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+mellitus+and+the+beta+cell:+The+last+ten+years.">Diabetes mellitus and the beta cell: The last ten years.</a> Cell. 148, 1160-1171 (2012).</li><li>Weir, G. C., Bonner-Weir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Five+stages+of+evolving+beta-cell+dysfunction+during+progression+to+diabetes.">Five stages of evolving beta-cell dysfunction during progression to diabetes.</a> Diabetes. 53, S16-S21 (2004).</li><li>Kjorholt, C., Akerfeldt, M. C., Biden, T. J., Laybutt, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+hyperglycemia,+independent+of+plasma+lipid+levels,+is+sufficient+for+the+loss+of+beta-cell+differentiation+and+secretory+function+in+the+db/db+mouse+model+of+diabetes.">Chronic hyperglycemia, independent of plasma lipid levels, is sufficient for the loss of beta-cell differentiation and secretory function in the db/db mouse model of diabetes.</a> Diabetes. 54, 2755-2763 (2005).</li><li>Wang, Y. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+type+2+diabetic+rodent+models.">Spontaneous type 2 diabetic rodent models.</a> Journal of Diabetes Research. , (2013).</li><li>Hummel, K. P., Dickie, M. M., Coleman, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetes+a+new+mutation+in+mouse.">Diabetes a new mutation in mouse.</a> Science. 153, 1127-1128 (1966).</li><li>Do, O. H., Low, J. T., Gaisano, H. Y., Thorn, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+secretory+deficit+in+islets+from+db/db+mice+is+mainly+due+to+a+loss+of+responding+beta+cells.">The secretory deficit in islets from db/db mice is mainly due to a loss of responding beta cells.</a> Diabetologia. 57, 1400-1409 (2014).</li><li>Low, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+principally+regulates+insulin+secretion+in+mouse+islets+by+controlling+the+numbers+of+granule+fusion+events+per+cell.">Glucose principally regulates insulin secretion in mouse islets by controlling the numbers of granule fusion events per cell.</a> Diabetologia. 56, 2629-2637 (2013).</li><li>Takahashi, N., Kishimoto, T., Nemoto, T., Kadowaki, T., Kasai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+pore+dynamics+and+insulin+granule+exocytosis+in+the+pancreatic+islet.">Fusion pore dynamics and insulin granule exocytosis in the pancreatic islet.</a> Science. 297, 1349-1352 (2002).</li><li>Low, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+secretion+from+beta+cells+in+intact+mouse+islets+is+targeted+towards+the+vasculature.">Insulin secretion from beta cells in intact mouse islets is targeted towards the vasculature.</a> Diabetologia. 57, 1655-1663 (2014).</li><li>Pologruto, T. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ScanImage:+flexible+software+for+operating+laser+scanning+microscopes.">ScanImage: flexible software for operating laser scanning microscopes.</a> Biomedical Engineering Online. 2, 13 (2003).</li></ol>

The authors declare they have no competing financial interests.

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，消化时间主要影响胰岛产量只有30秒的时间差，可以显着地影响质量。对于LIBERASE只，产量是可以接受的，但在消化有时会发生，我们认为，在加入胶原酶有助于消化结缔组织更好。 有若干可在胰岛已经分离之后进行可能的实验。胰岛可以固定在分离之后立即多聚甲醛或甲醇与用于免疫荧光染色，以确定感兴趣的10种蛋白的位置。胰岛可以分散在单细胞。这可以在胰岛分离的同一天或之后胰岛培养几天来完成。然后将单细胞铺板出来，这是该制剂已经作为支柱如膜片钳和全内反射显微技术。在该实验中测量胰岛素的分泌，以及用于活细胞2光子成像，我们已发现，胰岛需要在培养2天以得到再生的数据。什么是发生在文化不是很清楚，但被认为是一个周期从隔离和消化过程的恢复。 这里介绍的双光子技术使研究人员能够图像的胰岛素分泌过程在单个颗粒融合从完整的胰岛内的许多细胞中的水平。我们已发现，颗粒融合事件和它们的定时的数量是与量和胰岛素分泌8的时间曲线相一致。这表明该方法的检测的大多数胰岛素颗粒融合事件。鉴于在到定影颗粒染料项不是正在研究7,8检测，我们已经竭尽全力胰岛素颗粒融合事件进行对照实验证明β细胞胰岛素颗粒融合的具体方法</sup&gt;。然而，这种证据的一个组成部分是依赖于啮齿动物胰岛，其中β细胞富集的胰岛的芯的结构。人胰岛，其中不同的内分泌细胞分散在整个胰岛体积，所述的不同的结构将需要额外的方法来识别β细胞。 有许多的技术问题与双光子显微镜是通用于所有的成像方法。例如，我们仔细平衡试图获得足够的光线中的胰岛，使我们能够降低探测器的增益的对立因素，因此降低噪音，与把这么多的光中，我们造成细胞损伤胰岛。在我们的系统中，Pockels盒用来衰减脉冲激光（ 图3）;所有商业系统有某种机制来调节光的落在到试样的强度。 总之，仔细隔离分贝的组合/ Db胰岛和全胰岛2光子显微镜本新的机会来研究胰岛素分泌的方法，并确定在2型糖尿病的主要故障元件。

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。

<table border="0" cellpadding="0" cellspacing="0" width="675">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:173px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liberase TL 5mg</td>
			<td style="width:173px;">Roche</td>
			<td style="width:119px;">-5401020001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase type IV-1g</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">17104-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Histopaque 1077 500ml</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:119px;">10771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 Medium (10X) 1L</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:119px;">R1383</td>
			<td>to prepare isolation media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 (1X) 500ml</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">21870-076</td>
			<td>to prepare cultured media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin-Streptomycin 100ml</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>to prepare cultured media&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal bovine serum 500ml</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">10099-141</td>
			<td>to prepare cultured media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td style="width:173px;">Gibco-Life Technologies</td>
			<td style="width:119px;">11966-025</td>
			<td>to dilute the liberase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metamorph program</td>
			<td style="width:173px;">Molecular Devices, USA</td>
			<td style="width:119px;"></td>
			<td>to analyze the 2-photon images</td>
		</tr>
	</tbody>
</table>

leprdb mouse model of type 2 diabetes: pancreatic islet isolation and live-cell 2-photon imaging of intact islets

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。

胰岛产量和纯化对于一个正常的野生型小鼠，大约200胰岛预期。健康的胰岛看起来明亮，圆形并有光滑的边框。过消化隔离批次通常具有小和模糊胰岛而一个下消化批次具有较少的胰岛和腺泡细胞附着胰岛（ 图3）。糖尿病db / db小鼠，胰岛的收获量和外观取决于疾病进展具有更好的血糖小鼠具有更大，更明亮，更胰岛（高达300胰岛）相比，更坏的血糖小鼠具有较小的胰岛具有半透明的外观（在100胰岛） 2光子检测 2光子成像是一种间接测定法来测量胰岛素分泌以单一胰岛素颗粒融合的水平。响应于刺激，例如葡萄糖或高钾，胰岛素颗粒熔合质膜和细胞外染料SRB进入第Ë颗粒，这导致突然荧光斑点〜400nm的直径的外观（ 图5）。各种实验已经进行了验证该测定作为融合含有胰岛素的颗粒，如葡萄糖剂量依赖性增加的颗粒融合事件数目与胰岛素分泌的期望量，所述响应单元中的2的大小一致的的测量光子与β细胞的，由2光子和电子显微镜测得的类似颗粒直径，染料摄取和胰岛素的共定位染色8。 内记录时，响应于刺激识别和事件的像吻 - 运行或全融合的融合事件的位置，外观的时间，持续时间和类型的所有胞吐事件的特征（ 图5，6）。在融合过程中，这些特性可用于评估有价值<html>在2型糖尿病模型的颗粒融合的任何缺陷。我们使用这种方法以前的报告显示，在糖尿病的db / db胰岛缺陷为满颗粒融合的损失，而不是在单个颗粒融合7动力学的特性的变化。此外，我们表明，在降低胰岛素分泌的疾病的最大因素是在应答细胞7的数量的减少。 <img alt="图1" src="/files/ftp_upload/52632/52632fig1.jpg" /> 图1:夹紧和喷射的部位的图注入的酶的肝总管和胆囊管的交界处附近的胆总管。夹紧瓦尔特壶腹通过血管钳，以方便注射酶到胰管。 2632fig2.jpg"/&gt; 图2:在小鼠中的注入过程 。顶部图说明胆总管施加在瓦尔特壶腹血管钳内的针。下图显示了注射后LIBERASE灌注胰腺。 <img alt="图3" src="/files/ftp_upload/52632/52632fig3.jpg" /> 图3:野生型和的db / db胰岛典型图像（A）野生型胰岛。 （B）的db / db孤立小岛。 （C）在消化胰岛附带腺泡细胞（箭头）。 （ 四）在消化胰岛小和模糊的小岛（箭头）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52632/52632fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" sRC ="/文件/ ftp_upload / 52632 / 52632fig4.jpg"/&gt; 图4:订造2光子显微镜的主要组成部分 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52632/52632fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52632/52632fig5.jpg" /> 图5:为记录有2光子显微镜单一胰岛素颗粒融合事件的实施例左数据代表前（A）和后（B）一种胞吐事件（箭头）响应于15mM的外观葡萄糖刺激的细胞。 （C）表示与细胞外染料SRB的进入颗粒的SRB标记，当它与质膜融合。 （D）示出以上的胞吐事件的感兴趣区域的荧光强度以及sequentia此事件胞吐（比例尺1微米）的L-图像。图片来源做等人 2014年7修改。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52632/52632fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52632/52632fig6.jpg" /> 图6:双光子显微镜用SRB作为细胞外染料下的野生型和糖尿病的db / db胰岛的响应 。在这些实验中胰岛素颗粒融合事件响应于15mm的葡萄糖刺激被记录。黄色圆圈是胞吐事件的过程中记录的20分位点。图片来源做等 2014 7修改。

Watch this Scientific Journal Video about Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets at JoVE.com

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, ...

leprdb-mouse-model-type-2-diabetes-pancreatic-islet-isolation-live

Nanyang Technological University

Research

JoVE Journal

Medicine

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

9.4K 次观看.  The University of Queensland. 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.

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

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

从部分Pancreatectomized患者的人类胰岛的分离

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

科研

医学

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

人类胰岛染色协议

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Quantifying Glomerular Permeability of Fluorescent Macromolecules Using 2-Photon Microscopy in Munich Wistar Rats

Kidney diseases involving urinary loss of large essential macromolecules, such as serum albumin, have long been thought to be caused by alterations in the permeability barrier comprised of podocytes, vascular endothelial cells, and a basement membrane working in unison. Data from our laboratory using intravital 2-photon microscopy revealed a more permeable glomerular filtration barrier (GFB) than previously thought under physiologic conditions, with retrieval of filtered albumin occurring in an early subset of cells called proximal tubule cells (PTC)1,2,3.
Previous techniques used to study renal filtration and establishing the characteristic of the filtration barrier involved micropuncture of the lumen of these early tubular segments with sampling of the fluid content and analysis4. These studies determined albumin concentration in the luminal fluid to be virtually non-existent; corresponding closely to what is normally detected in the urine. However, characterization of dextran polymers with defined sizes by this technique revealed those of a size similar to serum albumin had higher levels in the tubular lumen and urine; suggesting increased permeability5.
Herein is a detailed outline of the technique used to directly visualize and quantify glomerular fluorescent albumin permeability in vivo. This method allows for detection of filtered albumin across the filtration barrier into Bowman's space (the initial chamber of urinary filtration); and also allows quantification of albumin reabsorption by proximal tubules and visualization of subsequent albumin transcytosis6. The absence of fluorescent albumin along later tubular segments en route to the bladder highlights the efficiency of the retrieval pathway in the earlier proximal tubule segments. Moreover, when this technique was applied to determine permeability of dextrans having a similar size to albumin virtually identical permeability values were reported2. These observations directly support the need to expand the focus of many proteinuric renal diseases to included alterations in proximal tubule cell reclamation.

A technique utilizing high resolution intavital 2-photon microscopy to directly visualize and quantify gloemrular filtration in surface glomeruli. This method allows for direct determination of permeability characteristics of macromolecules in both normal and diseased states.

利用双光子显微镜在慕尼黑Wistar大鼠量化肾小球通透性荧光大分子

Kidney  diseases involving urinary loss of large essential macromolecules, such as  serum albumin, have long been thought to be caused by alterations in ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

从手术标本的人类胰岛细胞激光显微切割的改进方法

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

分离，培养和成像胎儿胰腺细胞集群

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

原位胰腺癌小鼠模型的动态对比增强磁共振成像

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

通过胰管结扎手术损伤的小鼠胰腺作为模型内分泌和外重新规划和扩散

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

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 ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

用线 Myography 测定小鼠肠系膜动脉的等轴收缩力

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology have been developed and utilized in vivo, they are limited in terms of allowing single cell resolution and continuous longitudinal recordings. Because of the transparency of the cornea, the anterior chamber of the eye (ACE) in mice is well suited to study human and mouse pancreatic islet cell biology. Here is a description of how this approach can be used to perform continuous longitudinal recordings of grafting and revascularization of individual human islet grafts. Human islet grafts are inserted into the ACE, using NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mice as recipients. This allows for the investigation of the expansion of recipient versus donor cells and the contribution of recipient cells in promoting the encapsulation and vascularization of the graft. Further, a step-by-step approach for image analysis and quantification of the islet volume or segmented vasculature and islet capsule forming recipient cells is outlined.

The goal of this protocol is to continuously monitor the dynamics of the human pancreatic islet engraftment process and the contributing host versus donor cells. This is accomplished by transplanting human islets into the anterior chamber of the eye (ACE) of an NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mouse recipient followed by repeated 2-photon imaging.

前眼室人类胰岛移植和贡献宿主细胞的纵向维沃成像和定量

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology ...

瘦素受体

摘要

探索更多视频

此视频中的章节