这个工作是由来自美国，以色列边防军的赠款，以BS和EDSBS是分子遗传学的希尔达和塞西尔·刘易斯教授主持的一个义不容辞的责任。

注:涉及受试动物的程序获得批准的机构动物护理和使用委员会（IACUC）在魏茨曼科学研究所。 1，样品制备<ol><li>胰腺腺泡隔离<ol><li>在开始之前，请准备300毫升新鲜的Krebs-Ringer碳酸氢盐HEPES（KRBH）中。搭配140毫米的NaCl，4.7 mM的氯化钾，1.16毫米氯化镁2,1毫米氯化钙2，11毫米的葡萄糖，10毫米肝素钠。添加牛血清白蛋白（BSA）和0.1毫克/毫升大豆胰蛋白酶抑制剂（STI）（悬浮介质）。制备5 - 10毫升再悬浮的介质补充有胶原酶（〜0.75毫克/毫升）（消化培养基）。 </li><li>含氧的KRB介质〜30分钟。充氧后加入BSA，防止起泡。把悬浮和消化介质为37℃。注:补氧介质降低细胞的死亡率，并提高响应能力分泌。氧合的效果是明显的，至少6小时。 </li><lI&gt;牺牲鼠标颈椎脱位或CO 2窒息，根据机构的动物护理指南。将动物处死在它的后面，将其附加到夹层垫，清洁腹部表面用乙醇。 注意:为了避免污染，高压灭菌解剖工具和用于在使用前KRBH中的水。过滤器通过0.2微米的过滤器KRBH媒介。之前的胰腺切除冲洗清扫工具用70％的乙醇，并进行解剖上的清洁架。开展3级生物安全柜病毒感染。添加抗生素将O / N培养用培养基（见第4节）。 </li></ol></li><li>小鼠胰腺切除术<ol><li>切口广泛的腹部皮肤和皮下组织层。鉴定肝，胃，肠，胰腺和脾脏（ 图1）。注:在下面的步骤，请小心操作胰腺组织，避免用力过大，机械FORCE可防止自身消化的组织和细胞死亡。 </li><li>通过使用两个钝钳，拉出小肠。开始在小肠近端，从胃的出口附近。从肠中分离出胰腺和在体腔内保持它。一旦到达大肠，发现一个粉红色/白色结缔组织。不要与胰腺混淆。 </li><li>清除尽可能多的胰腺组织尽可能不收取脂肪或结缔组织。由脾胃分离，并切断腹壁血管下方（ 图1A-C）取出胰腺。 注意:由于胰腺腺泡从单一动物的产率是高的，就可以从收集所有的胰腺组织，以防止收集的其他组织避免。 </li></ol></li><li>胶原酶消化<ol><li>冲洗胰腺几次在悬浮介质中加热到37℃，并删除非胰腺其余部分组织。沉浸在〜5毫升预加热消化培养基的胰腺在20ml的闪烁瓶中。切碎胰腺1-3毫米的碎​​片。将它放置在恒温水浴（最好在〜100转的搅拌）。为了加速消化，吸取消化组织，上下每隔几分钟。 注:胶原酶消化的持续时间是主要依赖胶原酶浓度和对机械力施加在组织上的量。采用0.75毫克/毫升胶原酶约15分钟，并吸移该组织与5ml的聚苯乙烯吸管有2mm孔口每3 - 5分钟，产生10的腺泡 - 30个细胞。消化之后，悬挂出现浑浊含有肉眼可见的组织。由于胶原酶效率不同的商业类型和批次间的差异，决定了消化的时间经验。 </li><li>洗涤和过滤<ol><li>转移组织消化到50毫升管和终止挖estion加入45毫升悬浮介质中。通过粗过滤悬浮液（〜1毫米）金属网成新管。离心流过，在100×g下3分钟。 </li><li>弃去上清液并重新悬浮消化的胰腺在50ml悬浮介质中。通过70或100微米的尼龙网过滤悬浮液。离心机和以前一样。 </li><li>为了除去浮，损坏腺泡，弃去上清，重悬在胰腺组织中3 - 5毫升悬浮介质中。申请的胰腺腺泡1毫升在15ml锥形管中填充用7ml悬浮介质中。允许腺泡沉降2 - 3分钟。收集落户在以1毫升吸管的管底的腺泡，并重复这一步骤2 - 3次。 </li><li>查看光或解剖镜下胰腺腺泡来测试自己的病情。在图2中，观察隔离的纯度和损害后的细胞杜里造成吴隔离。立即使用这些腺泡或文化长达20小时。 </li></ol></li></ol></li></ol> 2，淀粉酶分泌测定注:淀粉酶分泌测定可作为胰腺腺泡分泌能力的全球措施。这是通过收集在该胰腺腺泡孵育培养基中，并确定该介质的淀粉酶的活性，通常是通过使用市售的试剂盒来实现的。 <ol><li>确定要检查实验条件的号码（ 如无刺激与刺激）。使用至少三次重复为每个条件。此外，添加一组重复，以确定细胞总淀粉含量（"总"的样品）和一组为中（"零"的样品）的淀粉酶活性。标记的多井板，其中该测定将进行的，并且两组微管对于每个重复的使用。 注:12或24孔板S可以是用于监测1和0.5ml腺泡悬浮液，分别。 </li><li>在30毫升KRBH悬浮培养基洗两次腺泡和在37℃下孵育它们在悬浮培养基中进行30分钟。到多孔板中添加促分泌的各10μl滴达到适当的终浓度。 </li><li>胰腺腺泡分泌到了一个两相的方式渐变回应。具有最佳价值的分泌浓度为50 - 100分胆囊收缩素（CCK）和卡巴的1μM（ 图3A）5。 </li><li>清洗和悬浮在悬浮介质中的腺泡。确保腺泡分布均匀的悬浮体的悬浮液，并等分试样等量入多孔板并进入"total&#39;标记的微管。孵育板30分钟，在37℃。 （最好在温和搅拌水浴（〜50 RPM））。 注:腺泡的确切数量可以experimen之间​​变化TS而应该在每次实验中重复类似。因此，腺泡悬浮液的精确等分是对于不同的重复和条件之间进行比较关键的。 </li><li>在培育期间，产生了&#39;总&#39;和&#39;零&#39;的样本。离心机的&#39;总&#39;微管几秒钟为5000×克，并从上清取出50微升到"zero&#39;标记的微管，并放置在4℃。裂解的腺泡的"总"的样品中添加的Triton X-100的微管，以达到1％的最终浓度。大力旋涡的微管。 注:小份的"总"复制类似实验的重复。裂解的腺泡，导致总淀粉含量的释放。或者，收集腺泡在每种条件和裂解在实验结束时，以确定每个重复的细胞含量。 </li><li>收集的大部分腺泡暂停íNTO第一组中的预标记的微管。离心微管几秒钟为5000×克。将上清液转移到第二组的预标记的微管。 </li><li>测定上层清液的淀粉酶活性用市售的试剂盒。计算淀粉酶的分泌的百分比。 注:这是非常重要的，在阅读其线性范围内淀粉酶的活性。目前淀粉酶的分泌淀粉酶的从初始总含量释放百分比的百分比。减去从各三份培养基的背景淀粉酶水平和由同样归初始总含量划分它。 </li></ol> 3，现场胰腺分泌成像来自胰腺腺泡分泌可以直接通过实时成像进行监控。使用各种荧光染料和传感器，实时成像允许分泌的表征在亚细胞分辨率。 <ol><li>准备新鲜的含氧悬浮介质，因为我n阶1.1并把它带到37℃。置的显微镜，如果配有温度控制器，将其调整到37℃。 </li><li>在30毫升悬浮介质的清洗腺泡细胞，并将其放置在玻璃或塑料滑梯。注意:如果亲脂性染料是要用于标签标记的亲脂性染料质膜，弄脏腺泡不超过5分钟，以防止内部的膜的标签通过内吞作用。在整个工作中，小心操作腺泡和避免过度的移液。 </li><li>一旦在显微镜图像腺泡的形态清晰，避免腺泡这​​显示基底泡或细胞内空泡的成像。添加促分泌逐滴或通过使用灌注腔，过程中或成像的开始之前立即。 注:高倍率镜头具有高数值孔径（ 如 ×63 / 1.4×100 / 1.4）的建议，观察亚细胞结构。 </li></ol> 4。 O /胰腺腺泡N个文化（备用细胞培养技术） 注:O 2 / N培养通常用于腺病毒感染。感染是在10 6 -10 7 PFU / ml的为9 -检查前16小时。 <ol><li>培养胰腺腺泡长达20小时。从一个单一的胰腺在25毫升培养基（DMEM所得悬浮腺泡补充有胎牛血清（FCS）（5％），钠丙酮酸盐（1％），抗生素（1％），L-谷氨酰胺（0.5％），牛血清白蛋白（10毫克/毫升）和STI（0.2毫克/毫升）中，并分成五个10厘米板。杂志征稿与病毒的腺泡在3级生物安全柜中。 </li><li>孵育胰腺腺泡在37℃，5％CO 2的潮湿空气。允许腺泡到后续检查3,8之前，在含氧的再悬浮介质中恢复30分钟。 </li></ol>

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The authors declare they have no competing financial interests.

除了 ​​在体外培养，为外分泌腺分泌的研究替代性设置包括从胰管液的活体显微镜和测量。活体显微镜结果表明在小鼠唾液腺10井进行操作。这个方法可以记录分泌实时的完整动物，但在其分辨率和由所述用于操纵外分泌组织被限制。通过从胰管收集体液评估分泌麻醉大鼠11来实现的。然而，由于胰管插管是极其困难的，这种方法很少使用。 这些设置相比，胰腺腺泡的体外培养是简单，快速，可用于监控和操纵细胞可及的手段，而不与完整的组织4,5的组织冲突。然而，这种设置有它的缺点。胰腺癌腺泡细胞是微妙的性质和变化培养条件非常敏感。由于细胞加载的蛋白水解酶，它们容易溶解和细胞死亡。其结果是，这种文化是短暂的，通常不会持续超过20小时。 它始终是最好辨认，并在产生解决问题。键的方法步骤是隔离完整胰腺腺泡无细胞损伤和死亡显著迹象。这样的迹象是很明显的，包括过度的细胞碎片和腺泡与基底泡或细胞内空泡。在淀粉酶分泌测定，细胞损伤将导致低刺激/非刺激分泌率。为了防止这样的损坏，建议轻轻处理的组织，加入BSA和​​STI的介质，并用氧气丰富它。例如，如果没有氧合酶，淀粉酶口罩的分泌的刺激作用的细胞过度放电（ 图图3C）。然而，由于外分泌组织的脆弱性，一些组织损伤几乎是不可避免的 - 这些制剂应被丢弃。 对于淀粉酶分泌测定，取上清液，其含有分泌的淀粉酶，但不含细胞，可以保持在4℃下进行1 - 2天。这上清应该再由商业试剂盒进行检测。许多可用的套件依赖人工淀粉酶底物的裂解和随后释放生色。因此，高淀粉酶浓度导致溶液的颜色变化更快。自分泌的淀粉酶将最终释放所有的发色团，从衬底，它必须按照随时间的反应，并只使用期间线性相位收集的数据。 胰腺腺泡实时成像可以为研究者一个令人沮丧的经历。一些细胞制剂，尤其是那些被感染的腺泡，可能不适合于实时成像由于腺泡破坏形态或水平的荧光传感器的不足。即使是在适当的准备，这可能需要一些时间来找到合适的标本。因而成像可能需要相当长的时间。 Lifeact-GFP，一架F-肌动蛋白探针，值得注意的是12。此工具允许我们遵循的F-肌动蛋白的动力学过程胰腺分泌长达16小时感染后，在分辨率的水平，可能不能达到固定的腺泡样品中。此外，采用单胞吐事件的Lifeact探头启用跟踪，如胰蛋白酶原囊泡进行肌动蛋白涂层前不久胞吐3,13。 统称掌握时，此处所描述的技术提供了研究者装置来评估胰腺分泌，并通过线束它来识别在该多步骤过程新颖的参与者。此外，考虑到胰腺和其他外分泌器官的结构和功能相似性，这些技术可以应用于其他外分泌组织，以及3。

外分泌胰腺构成了大部分的哺乳动物胰腺肿块，并致力于生产消化酶1的分泌。外分泌胰腺腺泡中，该功能单元是上皮细胞形成及股关节腔的集群。当激素或神经元的刺激，囊泡充满酶运输到顶端细胞表面，保险丝用它排出它们的内容到管腔1-3。所述分泌的酶，然后通过聚结组导管进入小肠，在那里它们催化的食物分解成的营养成分被排出。 此视频演示如何完好腺泡从整个胰腺被隔离，以及如何其分泌能力进行评估。采用这种设置，胰腺分泌是通过测量淀粉酶刺激后的被释放的相对量进行定量。或者，分泌可活通过使用不同的成像传感器和染料。 胰腺腺泡的离体的设置是有利的，因为分离的腺泡，其保留了完整的胰腺的许多特征，可以被操纵和监控的相比更容易在整个动物4-6。这种设置最初是在上世纪70年代，是因为延长了几个外分泌组织的研究，如唾液和乳腺3-7。值得注意的是，它允许分泌的与这些极化上皮细胞的复杂的细胞组织的干扰最小的研究。

<table><tbody><tr><td>ICR mice</td><td>Harlan</td><td></td><td>Any strain will do</td></tr><tr><td>HBSS</td><td>Sigma Aldrich</td><td>H8264</td><td>Can substitude for KRB</td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A7906</td><td>Fraction V</td></tr><tr><td>Trypsin inhibitor</td><td>Sigma Aldrich</td><td>T9003</td><td></td></tr><tr><td>Collagenase XI&amp;nbsp;</td><td>Sigma Aldrich</td><td>C0130</td><td></td></tr><tr><td>Nylon mesh</td><td>BD Falcon</td><td>352360</td><td>70-100&amp;micro;m</td></tr><tr><td>Amylase infinity reagent&amp;nbsp;</td><td>Thermo</td><td>TR25421</td><td></td></tr><tr><td>CCK</td><td>Sigma Aldrich</td><td>C2175</td><td></td></tr><tr><td>FM4-64</td><td>Lifetechnologies</td><td>F34653</td><td>Diluted to 16&amp;micro;M</td></tr><tr><td>20mL scintillation vial</td><td>Sigma Aldrich</td><td>Z190527</td><td></td></tr><tr><td>DeltaVision system&amp;nbsp;</td><td>Applied Precision</td><td></td><td>Consisting of an inverted microscope IX71 equipped with 60x/1.4 or 100x/1.3 objectives (Olympus)</td></tr><tr><td>Zeiss 510 or 710 confocal microscope</td><td>Zeiss</td><td></td><td>60x/1.4 or 100x/1.4 objectives&amp;nbsp;</td></tr><tr><td>Ultra Microplate reader</td><td>BioTek</td><td>ELx808</td><td></td></tr></tbody></table>

assessing the secretory capacity of pancreatic acinar cells

胰腺腺泡细胞产生和分泌的消化酶。这些细胞被组织成一个集群形成和股关节腔内。这项工作表明了这些细胞的分泌能力可以通过分离的腺泡的培养来评估。该设置是有利的，因为分离的腺泡，其保留了完整的胰腺外分泌的许多特征可被操纵和监控的相比更容易在整个动物。胰腺腺泡适当隔离是一个关键要求，以使体外培养将代表腺泡体内自然。该协议演示了如何从小鼠胰腺分离完好腺泡。随后，两个互补的方法来评估胰腺分泌呈现。淀粉酶的分泌试验作为一个全球性的措施，而胰腺分泌的直接成像可以分泌在亚细胞分辨率的表征。总的来说，所述技术presen泰德在这里让实验广谱学习外分泌。

通过透射光观察时进行了适当分离的胰腺腺泡显示一个刻板的形态。其基底领域应该会出现一轮且无泡的。顶域是由几百个分泌囊泡包围并显示在颜色（ 图2A，B）的更暗。细胞核位于基底的水泡区域。细胞碎片和内分泌胰腺，其可以在腺泡隔离（ 图2C，D）的早期阶段被检测到的胰腺导管系统和部件，应该是从最后腺泡悬浮缺席（ 图2A，B）。隔离不当，可能会导致产生基底泡和细胞破裂而排出的消化酶进入培养基（ 图2E，F）中。 从胰腺腺泡淀粉酶释放描绘了一个双相曲线时对分泌的浓度（ 图3A）作图。 W¯¯关于初始含量为5％ithout刺激过程中温育30分钟时释放。刺激后，这个数目是由多达5倍的升高。相比于新鲜分离的腺泡腺泡其培养的O / N，显示刺激与基淀粉酶的释放比率较低。重要的是，被感染腺病毒培养腺泡显示高基淀粉酶的分泌和刺激敏感的分泌（ 图3B，C）。因此，这是重要的感染控制腺泡具有相似的剂量控制病毒。 腺泡实时成像应该能清楚地识别腺泡细胞的基底和心尖域。亲脂性染料可有助于为窄顶域和细胞的横向和基底外侧的方面（ 图4）之间进行区分。根据生理刺激，分泌专门定向到顶端面1,9。使用F-肌动蛋白探针Lifeact-GFP的允许分泌囊泡的活可视化，因为胰蛋白酶原的囊泡进行的肌动蛋白涂覆前不久，胞吐作用（ 图4）。 <img alt="图1" fo:content-width="4in" src="/files/ftp_upload/51799/51799fig1highres.jpg" width="400" /> 图1:本地化胰腺和邻近器官。 （AC）的腹部皮肤和皮下组织层（A）的切口后，胰腺和邻近器官，才从邻近器官胰腺分离，（B）和（C）后，分别<a href="https://www.jove.com/files/ftp_upload/51799/51799fig1large.jpg" target="_blank">请点击这里查看大图这个数字。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51799/51799fig2highres.jpg" width="500" /> 图2分离胰腺腺泡。 （A）和（B）胰腺腺泡分离出正常显示刻板的形态;其基底外侧结构域出现圆形和不含泡的，而其顶端结构域是由几百个分泌囊泡包围，并显示在彩色（箭头）较暗（C），并可以被检测到（D）的细胞碎片和部件的胰腺导管系统第在腺泡隔离早期阶段（箭头指向的外分泌管）（E）和（F）的不正确的隔离导致产生基底泡（E，箭头），和细胞破碎，其放电的消化酶到培养基中（楼箭头）。标尺代表100微米。 <a href="https://www.jove.com/files/ftp_upload/51799/51799fig2large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> </P&gt; <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51799/51799fig3highres.jpg" width="500" /> 图3:测量从孤立腺泡淀粉酶的释放，来自胰腺腺泡（A）淀粉酶的释放描绘了双相曲线，当对一个分泌的浓度作图。腺泡分离并留下来恢复对刺激胆囊收缩素（CCK）的指定浓度之前30分钟。结果代表平均值和SEM的三个独立的实验（B）腺泡这 ​​是相比于新分离培养腺泡O / N，显示刺激与基淀粉酶的释放比率较低。被感染腺病毒培养腺泡显示高基淀粉酶的分泌和刺激敏感的分泌。（C）非充氧培养条件导致过度迪scharge淀粉酶的分泌的掩盖了刺激作用。 <img alt="图4" fo:content-width="6in" src="/files/ftp_upload/51799/51799fig4highres.jpg" width="600" /> 图4胰腺分泌的现场影像。现场刺激腺泡细胞，表达Lifeact-GFP（洛杉矶，绿，灰），并沾上了亲脂性染料FM4-64（红色）。使用Lifeact探头允许的肌动蛋白包被的囊泡（箭头）单融合事件的现场可视化。细胞感染O 2 / N与腺病毒Lifeact-GFP（广告，Lifeact-GFP）染色和成像开始前短暂的刺激与100分的CCK。比例尺条为5微米。 <a href="https://www.jove.com/files/ftp_upload/51799/51799fig4large.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about Assessing the Secretory Capacity of Pancreatic Acinar Cells at JoVE.com

Assessing the Secretory Capacity of Pancreatic Acinar Cells

孤立的胰腺腺泡保留其在体内的形态和活动，并提供强大的方式进行监控和操作的分泌。这项工作表明如何腺泡可以从小鼠胰腺中分离出来，并且其分泌能力如何进行评估。

评估胰腺腺泡细胞的分泌能力

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work ...

assessing-the-secretory-capacity-of-pancreatic-acinar-cells

Nanyang Technological University

Research

JoVE Journal

Biology

16.6K 次观看.  Weizmann Institute of Science. 孤立的胰腺腺泡保留其在体内的形态和活动，并提供强大的方式进行监控和操作的分泌。这项工作表明如何腺泡可以从小鼠胰腺中分离出来，并且其分泌能力如何进行评估。 

视频: Assessing the Secretory Capacity of Pancreatic Acinar Cells

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.

Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.

三维细胞培养中胰腺肌囊内增生的模拟与操纵

The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires ...

科研

癌症研究

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function.
Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions.
Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.

Transcellular protein interactions are important determinants of pancreatic beta-cell function. Detailed here is a method&#x2014;adapted from a coculture model of synaptogenesis&#x2014;for investigating how specific transmembrane proteins influence insulin secretion. Transfected HEK293 cells express proteins of interest; beta cells do not need to be transfected or otherwise directly perturbed.

共培养影响胰腺β细胞的胰岛素分泌细胞外蛋白质相互作用分析

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

医学

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those obtained with whole pancreas transplantation. The protocols currently used for human islet isolation are under scrutiny because they are based on the enzymatic digestion of the organ, whereby the pancreas is demolished, its connections to the body are lost and islets are irreversibly damaged. Islet damage is characterized by critical factors such as the destruction of the extracellular matrix (ECM), which represents the 3D framework of the islet niche and whose loss is incompatible with islet euphysiology. Researchers are proposing the use of ECM-based scaffolds derived from the mammalian pancreas to address this problem and ultimately improve islet viability, function, and lifespan. Currently available methods to obtain such scaffolds are harsh because they are largely detergent based. Thus, we propose a new, detergent-free method that creates less ECM damage and can preserve critical components of pancreatic ECM. The results show that the newly developed decellularization protocol allowed the achievement of complete DNA clearance while the ECM components were retained. The ECM obtained was tested for cytotoxicity and encapsulated with human pancreatic islets which showed a positive cellular behavior with insulin secretion when stimulated with glucose challenge. Collectively, we propose a new method for the decellularization of the human pancreas without the use of conventional ionic and non-ionic chemical detergents. This protocol and the ECM obtained with it could be of use for both in vitro and in vivo applications.

<meta charset="utf8"></head><body>人胰腺的无去污剂脱细胞化，用于可溶性细胞外基质 ECM 生产

The protocol described in this manuscript explains the steps for the fabrication of a soluble extracellular matrix (ECM) from the human pancreas. The solubilized ECM powder obtained through this protocol may be used for the recapitulation of pancreatic islets&#8217; microenvironment in vitro and, potentially, in vivo settings.

人胰腺的无去垢去细胞化，用于可溶性细胞外基质 （ECM） 生产

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those ...

生物工程

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 ...

生物学

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

制备胰腺腺泡细胞钙成像的目的，细胞损伤的测量，以及腺病毒感染

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

小鼠原代胰腺腺泡细胞的分离和培养

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

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

通过腹腔注射药物诱导小鼠胰腺损伤

Preparing a Mice Model of Severe Acute Pancreatitis via a Combination of Caerulein and Lipopolysaccharide Intraperitoneal Injection

The treatment of severe acute pancreatitis (SAP), with high mortality rates, poses a significant clinical challenge. Investigating the pathological changes associated with SAP using animal models can aid in identifying potential therapeutic targets and exploring novel treatment approaches. Previous studies primarily induced pancreatic injury through retrograde bile duct injection of sodium taviaurocholate, but the impact of surgical damage on the quality of the animal model remains unclear. In this study, we employed various frequencies of intraperitoneal Caerulein injections combined with different doses of LPS to induce pancreatic injury in C57BL/6J mice and compared the extent of injury across five intraperitoneal injection protocols. Regarding inducing acute pancreatitis in mice, an intraperitoneal injection protocol is proposed that results in a mortality rate as high as 80% within 5 days. Specifically, mice received ten daily intraperitoneal injections of Caerulein (50 &#181;g/kg), followed by an injection of LPS (15 mg/kg) one hour after the last Caerulein administration. By adjusting the frequency and dosage of injected medications, one can manipulate the severity of pancreatic injury effectively. This model exhibits strong controllability and has a short replication cycle, making it feasible for completion by a single researcher without requiring expensive equipment. It conveniently and accurately simulates key disease characteristics observed in human SAP while demonstrating a high degree of reproducibility.

作者聚焦:确定开发小鼠严重急性胰腺炎新疗法的治疗靶点

Intraperitoneal drug administration is a safe and effective non-invasive approach for inducing pancreatic injury. This study compared five distinct intraperitoneal injection protocols on mice to induce varying degrees of pancreatic injury and established a model of severe pancreatic injury to investigate the pathological changes and treatment strategies for severe acute pancreatitis (SAP).

通过 Caerulein 和 Lipopolysaccharide 联合腹腔注射制备严重急性胰腺炎小鼠模型

The treatment of severe acute pancreatitis (SAP), with high mortality rates, poses a significant clinical challenge. Investigating the pathological ...

评估胰腺腺泡细胞的分泌能力

摘要

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三维细胞培养中胰腺肌囊内增生的模拟与操纵

共培养影响胰腺β细胞的胰岛素分泌细胞外蛋白质相互作用分析

人胰腺的无去垢去细胞化，用于可溶性细胞外基质（ECM）生产

人胰腺的无去垢去细胞化，用于可溶性细胞外基质（ECM）生产

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

制备胰腺腺泡细胞钙成像的目的，细胞损伤的测量，以及腺病毒感染

小鼠原代胰腺腺泡细胞的分离和培养

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

通过 Caerulein 和 Lipopolysaccharide 联合腹腔注射制备严重急性胰腺炎小鼠模型

通过 Caerulein 和 Lipopolysaccharide 联合腹腔注射制备严重急性胰腺炎小鼠模型

评估胰腺腺泡细胞的分泌能力

摘要

探索更多视频

三维细胞培养中胰腺肌囊内增生的模拟与操纵

共培养影响胰腺β细胞的胰岛素分泌细胞外蛋白质相互作用分析

人胰腺的无去垢去细胞化，用于可溶性细胞外基质 （ECM） 生产

人胰腺的无去垢去细胞化，用于可溶性细胞外基质 （ECM） 生产

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

制备胰腺腺泡细胞钙成像的目的，细胞损伤的测量，以及腺病毒感染

小鼠原代胰腺腺泡细胞的分离和培养

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

通过 Caerulein 和 Lipopolysaccharide 联合腹腔注射制备严重急性胰腺炎小鼠模型

通过 Caerulein 和 Lipopolysaccharide 联合腹腔注射制备严重急性胰腺炎小鼠模型

人胰腺的无去垢去细胞化，用于可溶性细胞外基质（ECM）生产

人胰腺的无去垢去细胞化，用于可溶性细胞外基质（ECM）生产