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

<ol>
	<li>Cryer, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoglycaemia:+the+limiting+factor+in+the+glycaemic+management+of+Type+I+and+Type+II+diabetes.">Hypoglycaemia: the limiting factor in the glycaemic management of Type I and Type II diabetes.</a> Diabetologia. 45, 937-948 (2002).</li><li>Alberti, K. G., Zimmet, P. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition,+diagnosis+and+classification+of+diabetes+mellitus+and+its+complications.+Part+1:+diagnosis+and+classification+of+diabetes+mellitus+provisional+report+of+a+WHO+consultation.">Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation.</a> Diabet Med. 15, 539-553 (1998).</li><li>Muoio, D. M., Newgard, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+disease:+molecular+and+metabolic+mechanisms+of+insulin+resistance+and+beta-cell+failure+in+type+2+diabetes.">Mechanisms of disease: molecular and metabolic mechanisms of insulin resistance and beta-cell failure in type 2 diabetes.</a> Nat Rev Mol Cell Biol. 9, 193-205 (2008).</li><li>Kelly, C., McClenaghan, N. H., Flatt, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+islet+structure+and+cellular+interactions+in+the+control+of+insulin+secretion.">Role of islet structure and cellular interactions in the control of insulin secretion.</a> Islets. 3, 41-47 (2011).</li><li>Meda, P., Halban, P., Perrelet, A., Renold, A. E., Orci, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junction+development+is+correlated+with+insulin+content+in+the+pancreatic+B+cell.">Gap junction development is correlated with insulin content in the pancreatic B cell.</a> Science. 209, 1026-1028 (1980).</li><li>Hohmeier, H. E., 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=Isolation+of+INS-1-derived+cell+lines+with+robust+ATP-sensitive+K++channel-dependent+and+-independent+glucose-stimulated+insulin+secretion.">Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion.</a> Diabetes. 49, 424-430 (2000).</li><li>Hohmeier, H. E., Newgard, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lines+derived+from+pancreatic+islets.">Cell lines derived from pancreatic islets.</a> Mol Cell Endocrinol. 228, 121-128 (2004).</li><li>Field, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+insulin+by+liver.">Extraction of insulin by liver.</a> Annu Rev Med. 24, 309-314 (1973).</li><li>Emmanouel, D. S., 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=Glucagon+metabolism+in+the+rat.">Glucagon metabolism in the rat.</a> J Clin Invest. 62, 6-13 (1978).</li><li>Kimple, M. E., 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=Deletion+of+GalphaZ+protein+protects+against+diet-induced+glucose+intolerance+via+expansion+of+beta-cell+mass.">Deletion of GalphaZ protein protects against diet-induced glucose intolerance via expansion of beta-cell mass.</a> J Biol Chem. 287, 20344-20355 (2012).</li><li>Rabaglia, M. E., 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=Alpha-Ketoisocaproate-induced+hypersecretion+of+insulin+by+islets+from+diabetes-susceptible+mice.">Alpha-Ketoisocaproate-induced hypersecretion of insulin by islets from diabetes-susceptible mice.</a> Am J Physiol Endocrinol Metab. 289, 218-224 (2005).</li><li>Collins, S. C., Salehi, A., Eliasson, L., Olofsson, C. S., 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=Long-term+exposure+of+mouse+pancreatic+islets+to+oleate+or+palmitate+results+in+reduced+glucose+induced+somatostatin+and+oversecretion+of+glucagon.">Long-term exposure of mouse pancreatic islets to oleate or palmitate results in reduced glucose induced somatostatin and oversecretion of glucagon.</a> Diabetologia. 51, 1689-1693 (2008).</li><li>Fueger, P. T., Hernandez, A. M., Chen, Y. C., Colvin, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+Replication+and+Beta+Cell+Function+in+Adenovirally-transduced+Isolated+Rodent+Islets.">Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets.</a> J Vis Exp. , (2012).</li><li>Szot, G. L., Koudria, P., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+pancreatic+islet+isolation.">Murine pancreatic islet isolation.</a> J Vis Exp. , (2007).</li><li>Szot, G. L., Koudria, P., Bluestone, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+pancreatic+islets+into+the+kidney+capsule+of+diabetic+mice.">Transplantation of pancreatic islets into the kidney capsule of diabetic mice.</a> J Vis Exp. , (2007).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> J Vis Exp. , (2011).</li><li>Kimple, M. E., 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=A+role+for+G(z)+in+pancreatic+islet+beta-cell+biology.">A role for G(z) in pancreatic islet beta-cell biology.</a> J Biol Chem. 280, 31708-31713 (2005).</li><li>Shaw, J. E., Sicree, R. A., Zimmet, P. Z. <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+the+prevalence+of+diabetes+for+2010+and+2030.">Global estimates of the prevalence of diabetes for 2010 and 2030.</a> Diabetes Res Clin Pract. 87, 4-14 (2010).</li><li>Kirstetter, P., Lagneau, F., Lucas, O., Krupa, Y., Marty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+endothelium+in+the+modulation+of+isoflurane-induced+vasodilatation+in+rat+thoracic+aorta.">Role of endothelium in the modulation of isoflurane-induced vasodilatation in rat thoracic aorta.</a> Br J Anaesth. 79, 84-87 (1997).</li><li>Brown, E. T., Umino, Y., Loi, T., Solessio, E., Barlow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+can+cause+sustained+hyperglycemia+in+C57/BL6J+mice.">Anesthesia can cause sustained hyperglycemia in C57/BL6J mice.</a> Vis Neurosci. 22, 615-618 (2005).</li><li>Vaupel, D. B., McCoun, D., Cone, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phencyclidine+analogs+and+precursors:+rotarod+and+lethal+dose+studies+in+the+mouse.">Phencyclidine analogs and precursors: rotarod and lethal dose studies in the mouse.</a> J Pharmacol Exp Ther. 230, 20-27 (1984).</li><li>Davis, D. B., 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=FoxM1+is+up-regulated+by+obesity+and+stimulates+beta-cell+proliferation.">FoxM1 is up-regulated by obesity and stimulates beta-cell proliferation.</a> Mol Endocrinol. 24, 1822-1834 (2010).</li><li>Dionne, K. E., Colton, C. K., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+hypoxia+on+insulin+secretion+by+isolated+rat+and+canine+islets+of+Langerhans.">Effect of hypoxia on insulin secretion by isolated rat and canine islets of Langerhans.</a> Diabetes. 42, 12-21 (1993).</li><li>Linetsky, E., 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=Improved+human+islet+isolation+using+a+new+enzyme+blend,+liberase.">Improved human islet isolation using a new enzyme blend, liberase.</a> Diabetes. 46, 1120-1123 (1997).</li><li>Vaithilingam, V., Sundaram, G., Tuch, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+cell+transplantation.">Islet cell transplantation.</a> Curr Opin Organ Transplant. 13, 633-638 (2008).</li><li>Brandhorst, H., 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=Large-scale+comparison+of+Liberase+HI+and+collagenase+NB1+utilized+for+human+islet+isolation.">Large-scale comparison of Liberase HI and collagenase NB1 utilized for human islet isolation.</a> Cell Transplant. 19, 3-8 (2010).</li></ol>

作者什么都没有透露。

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

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

<table><tbody><tr><td>Collagenase from &lt;em&gt;Clostridium histolyticum&lt;/em&gt; suitable for isolating active islets</td><td>Sigma-Aldrich</td><td>C7657</td><td></td></tr><tr><td>Ficoll 400</td><td>Sigma-Aldrich</td><td>F9378</td><td></td></tr><tr><td>Hanks Balanced Salt Solution 10X</td><td>Invitrogen (Gibco)</td><td>14065-056</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>RPMI 1640 (powder)</td><td>Invitrogen (Gibco)</td><td>31800-022</td><td></td></tr><tr><td>Albumin from Bovine Serum (BSA)</td><td>Sigma-Aldrich</td><td>A7888</td><td></td></tr><tr><td>3/0 Silk Suture Thread</td><td>Fine Science Tools</td><td>18020-30</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-10</td><td></td></tr><tr><td>0.8 mm Forceps&amp;nbsp;&amp;nbsp;</td><td>Fine Science Tools</td><td>11050-10</td><td></td></tr><tr><td>Curved Scissors</td><td>Fine Science Tools</td><td>14061-10</td><td></td></tr><tr><td>Vannas-T&amp;uuml;bingen Spring Scissors - Straight/Sharp/8.5 cm/5 mm Cutting Edge</td><td>Fine Science Tools</td><td>15003-08</td><td></td></tr><tr><td>Dissecting Scissors</td><td>Fine Science Tools</td><td>14002-14</td><td></td></tr><tr><td>5 ml BD Luer-Lok Syringe</td><td>BD</td><td>309646</td><td></td></tr><tr><td>1 ml BD syringe</td><td>BD</td><td>309628</td><td></td></tr><tr><td>30 G BD Needle 1/2&quot; Length</td><td>BD</td><td>305106</td><td></td></tr><tr><td>27 G BD Needle 1/2&quot; Length</td><td>BD</td><td>305109</td><td></td></tr><tr><td>Sharpening Stone</td><td>Fine Science Tools</td><td>29008-01</td><td></td></tr><tr><td>2-2-2-tribromoethanol</td><td>Sigma-Aldrich</td><td>T48402-25G</td><td></td></tr><tr><td>2-methyl-2-butanol</td><td>Sigma-Aldrich</td><td>240486-100mL</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>Monopotassium Phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>P0662</td><td></td></tr><tr><td>Sodium Bicarbonate (NaCHO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S6014</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;#183;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>C3881</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;#183;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M9397</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Invitrogen (Gibco)</td><td>15140-122</td><td></td></tr><tr><td>Heat Inactivated Fetal Bovine Serum (H.I. FBS)</td><td>Fisher Scientific</td><td>SH30088.03HI</td><td></td></tr><tr><td>3-Isobutyl-1-methylxanthine (IBMX)</td><td>Sigma-Aldrich</td><td>5879-100MG</td><td></td></tr></tbody></table>

a method for mouse pancreatic islet isolation and intracellular camp determination

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

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

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

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

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

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

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

a-method-for-mouse-pancreatic-islet-isolation-intracellular-camp

Research

JoVE Journal

Biology

65.1K 次观看.  University of Wisconsin-Madison. 含量测定采用胰岛分离的小鼠胰岛体外 β细胞功能是在糖尿病病理生理学和治疗学研究的一个重要组成部分。虽然许多下游应用的情况下，该协议特别描述作为必要参数确定的β-细胞功能的细胞内环磷酸腺苷（cAMP）的测定。 

视频: A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

小鼠胰岛的分离和包膜下肾移植的方法

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

科研

医学

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

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, ...

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

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 the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced &#946; cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the &#946; cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in islet cells. This method should be useful for screening diabetogenic xenobiotics. In addition, the cultured islet cells may also be adapted for in vitro analysis of chemical-induced toxicity in pancreatic cells.

Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

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 ...

免疫与感染

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

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

小鼠胰岛的朗格汉斯的分离纯化的胶原酶和中性蛋白酶结合使用

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

生物学

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

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

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

利用邻近结扎测定法在人类胰腺β细胞中原位内源性米托巴结物的可视化

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

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

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

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

胰腺胰岛与小鼠分离的简单高效协议

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

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

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

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

小鼠胰腺胰岛细胞亚群中的钙动力学成像

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

<meta charset="utf8"></head><body>简化用于疾病研究的小鼠胰腺内皮细胞提取

Isolation of Mouse Pancreatic Endothelial Cells

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and glucagon, as well as digestive enzymes. The pancreas is also a highly vascularized organ, a feature facilitated by the intricate network of pancreatic capillaries. This extensive capillary network is made up of highly fenestrated endothelial cells (ECs) important for pancreas development and function. Accordingly, the dysfunction of ECs can contribute to that of the pancreas in diseases like diabetes and cancer. Thus, researching the function of pancreatic ECs (pECs) is important not only for understanding pancreas biology but also for developing its pathologies. Mouse models are valuable tools to study metabolic and cardiovascular diseases. However, there has not been an established protocol with sufficient details described for the isolation of mouse pECs due to the relatively small population of ECs and the abundant digestive enzymes potentially released from the acinar tissue that can lead to cell damage and, thus, low yield. To address these challenges, we devised a protocol to enrich and recover mouse pECs, combining gentle physical and chemical dissociation and antibody-mediated selection. The protocol presented here provides a robust method to extract intact and viable ECs from the whole mouse pancreas. This protocol is suitable for multiple downstream assays and may be applied to various mouse models.

<meta charset="utf8"></head><body>小鼠胰腺内皮细胞的分离

This protocol describes the isolation of mouse endothelial cells from whole pancreas.

小鼠胰腺内皮细胞的分离

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and ...

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

摘要

探索更多视频

此视频中的章节

小鼠胰岛的分离和包膜下肾移植的方法

小鼠胰腺内分泌细胞的单细胞 Transcriptomic 分析

瘦素受体

小鼠胰岛细胞筛选致糖尿病环境化学物质的研究

小鼠胰岛的朗格汉斯的分离纯化的胶原酶和中性蛋白酶结合使用

利用邻近结扎测定法在人类胰腺β细胞中原位内源性米托巴结物的可视化

胰腺胰岛与小鼠分离的简单高效协议

小鼠胰腺胰岛细胞亚群中的钙动力学成像

小鼠胰腺内皮细胞的分离

小鼠胰腺内皮细胞的分离