Name: a method for murine islet isolation and subcapsular kidney transplantation
Uploaded: 2011-04-13T11:00:00
Duration: 17 min 42 s
Description: Watch this Scientific Journal Video about A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation at JoVE.com

这项工作是由RO1 DK064938（TH）的支持。

<ol>
	<li>Shapiro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+transplantation+in+seven+patients+with+type+1+diabetes+mellitus+using+a+glucocorticoid-free+immunosuppressive+regimen.">Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.</a> N Engl J Med. 343, 230-238 (2000).</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=Islet+transplantation+as+a+treatment+for+diabetes.">Islet transplantation as a treatment for diabetes.</a> J Am Optom Assoc. 69, 727-732 (1998).</li><li>Gangemi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+transplantation+for+brittle+type+1+diabetes:+the+UIC+protocol.">Islet transplantation for brittle type 1 diabetes: the UIC protocol.</a> Am J Transplant. 8, 10-1111 (2008).</li><li>Zmuda, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiency+of+ATF3,+an+adaptive-response+gene,+protects+islets+and+ameliorates+inflammation+in+a+syngenic+transplantation+model.">Deficiency of ATF3, an adaptive-response gene, protects islets and ameliorates inflammation in a syngenic transplantation model.</a> Diabetologia. , (2010).</li><li>Bottino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+Human+Islets+to+Isolation+Stress+and+the+Effect+of+Antioxidant+Treatment.">Response of Human Islets to Isolation Stress and the Effect of Antioxidant Treatment.</a> Diabetes. 53, 2559-2568 (2004).</li><li>Markmann, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+islet+cell+culture+in+vitro+at+24+degrees+C+on+graft+survival+and+MHC+antigen+expression.">The effect of islet cell culture in vitro at 24 degrees C on graft survival and MHC antigen expression.</a> Transplantation. 49, 272-277 (1990).</li><li>Terasaka, R., Lacy, P. E., Hauptfeld, V., Bucy, R. P., Davie, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cyclosporin-A,+low-temperature+culture,+and+anti-Ia+antibodies+on+prevention+of+rejection+of+rat+islet+allografts.">The effect of cyclosporin-A, low-temperature culture, and anti-Ia antibodies on prevention of rejection of rat islet allografts.</a> Diabetes. 35, 83-88 (1986).</li><li>Ricordi, C., Lacy, P. E., Sterbenz, K., Davie, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-temperature+culture+of+human+islets+or+in+vivo+treatment+with+L3T4+antibody+produces+a+marked+prolongation+of+islet+human-to-mouse+xenograft+survival.">Low-temperature culture of human islets or in vivo treatment with L3T4 antibody produces a marked prolongation of islet human-to-mouse xenograft survival.</a> Proc Natl Acad Sci U S A. 84, 8080-8084 (1987).</li><li>Schnedl, W. J., Ferber, S., Johnson, J. H., 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=STZ+transport+and+cytotoxicity.+Specific+enhancement+in+GLUT2-expressing+cells.">STZ transport and cytotoxicity. Specific enhancement in GLUT2-expressing cells.</a> Diabetes. 43, 1326-1333 (1994).</li><li>Eckhard, M., Brandhorst, D., Brandhorst, H., Brendel, M. D., Bretzel, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+in+osmolality+and+range+of+density+of+a+continuous+ficoll-sodium-diatrizoate+gradient+for+isopycnic+purification+of+isolated+human+islets.">Optimization in osmolality and range of density of a continuous ficoll-sodium-diatrizoate gradient for isopycnic purification of isolated human islets.</a> Transplantation Proceedings. 36, 2849-2854 (2004).</li><li>Gores, P. F., Rabe, F., Sutherland, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+survival+of+intraportal+versus+subrenal+capsular+transplanted+islet+allografts.">Prolonged survival of intraportal versus subrenal capsular transplanted islet allografts.</a> Transplantation. 43, 747-749 (1987).</li><li>Nasr, I. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testicular+Immune+Privilege+Promotes+Transplantation+Tolerance+by+Altering+the+Balance+between+Memory+and+Regulatory+T+Cells.">Testicular Immune Privilege Promotes Transplantation Tolerance by Altering the Balance between Memory and Regulatory T Cells.</a> J Immunol. 174, 6161-6168 (2005).</li><li>Merani, S., Toso, C., Emamaullee, J., Shapiro, A. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+implantation+site+for+pancreatic+islet+transplantation.">Optimal implantation site for pancreatic islet transplantation.</a> British Journal of Surgery. 95, 1449-1461 (2008).</li><li>Inoue, M., Maeno, T., Hatchell, 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=Survival+of+allografted+pancreatic+islets+in+the+subretinal+space+in+rats.">Survival of allografted pancreatic islets in the subretinal space in rats.</a> Ophthalmic Res. 35, 48-53 (2003).</li></ol>

在上面的章节中，我们描述了隔离和小鼠胰岛移植的详细步骤。在这里，我们简要地强调程序成功的关键步骤。 1。胰岛隔离关键的步骤是确定一个最佳的酶消化时间（1.4），定位在十二指肠，胆总管（2.2.3）开放的止血钳，后37 cannulating胆总管（2.3），胰腺剧烈摇晃° C精华（3.2），并拆除外分泌细胞污染（3.3.7和3.3.16）。这些步骤的质量，产量和纯度的胰岛的一个戏剧性的影响。也许是最具技术挑战的第一步是胆总管插管。在许多小鼠，胆总管可很难想象，由于周围的筋膜。因此，它是常见的，试图cannulate胆总管的筋膜组织的渗透管的结果。在这些情况下，Liberase开始，以填补周围结缔组织和不灌注胰腺。如果发生此种情况，停止的Liberase流动和重新定位针，使其渗透进入胆管管腔。通过实践，它是可以快速识别针放置的最佳位置，以不破坏易碎管的情况下完成插管。 2。胰岛移植关键步骤是STZ诱导的糖尿病（5.1）和胰岛的有效传递到包膜袋（5.3.14-5.3.16）。菌素是一种自然发生的葡萄糖类似物，是有选择性的胰岛素分泌细胞，胰岛9β-细胞毒性。然而，这种选择性的毒性发生在一个相对狭窄的浓度范围内。菌素高剂量可以导致高血糖的情况下迅速（2-3天）杀伤力的事实证明了这一点。因此，应采取应变和任何大规模实验开始前正在使用的小鼠的年龄，以确定最佳剂量链脲佐菌素。胰岛实物交割的包膜囊也是一个关键的一步。为了达成一致的结果，这是势在必行，以尽量减少损失胰岛期间及其运载工具。是否有缺陷或在肾囊囊的眼泪，或如果注入的体积过大，可能发生这种情况。温柔细心操作，避免使用光滑的玻璃探针准备包膜囊囊的损害，可以。此外，注射量可以减少小心去除上清液中的步骤5.3.6.3。如果上清液去除沉淀胰岛水平，通常有足够的空间，在包膜袋放在400至500胰岛多。但是，如果注射量太大，胰岛更容易泄漏出来的袋子，在移植过程中，影响实验的可重复性。 在这个协议中，它是可以修改的一些步骤，而且还能达到令人满意的成果。这些措施包括以下内容：（一），（b）为胰腺酶的不同方法，使用不同的酶消化胰腺（C）使用一个连续的聚蔗糖 - 钠泛影葡胺（FSD）梯度而不是单一密度Histopaque1077一步，和（D）胰岛移植肾subcapsule以外的地点。 <ol class="lalpha"><li>在这个协议中，Liberase是用来削弱内的胰腺细胞与细胞接触。 Liberase本质上是一种高度纯化的胰腺中的胰岛释放已被证明有用胶原酶的混合物。然而，少纯化的胶原酶能够完成了类似的结果。因此，它可以使用不同的商业用酶制剂，消化胰腺，只要消化条件进行了优化，实现高胰岛质量，产量和纯度。 </li><li>它也可能无导管灌注胰腺酶消化。 Liberase通过胆总管交付允许胰腺表面面积最大的酶接触。甚至更多的胰腺和更完整的胰岛释放的消化这个结果。然而，其他步骤增加胰腺酶的表面积，可以使用。例如，切碎之前在Liberase孵化或直接通过胰腺囊注射Liberase胰腺可用于孤立的小岛。然而，没有Liberase交付的另一种方法是通过胆总管灌注的有效。因此，切碎或直接注射应保留作为最后的手段，如果胆总管损坏，而不是perfusable。 </li><li>从外分泌细胞的分离胰岛的另一种方法涉及使用了消防处的梯度，而不是Histopaque1077 10。从本议定书的外分泌细胞胰岛的高效分离是由于两种类型的细胞之间的密度差的一部分。 Histopaque1077已为1.0771克/毫升的密度在25 ° C。在此温度下，Histopaque是较密集的小岛，但比外分泌细胞密集。因此，在离心，Histopaque1077颗粒的外分泌细胞的力量，同时解除胰岛表面。密度与任何其他物质，可以独立于外分泌细胞胰岛的原则，可以在此步骤中使用和消防处的梯度是一个这样的物质。 </li><li>关于胰岛移植的位置，在本议定书中所使用的肾囊是几个可能的地点之一。而肾subcapsule是最常报道的网站在小鼠体内移植，移植其他网站都被发现是有效的，它们包括肝门静脉，视网膜下腔，睾丸，附睾脂肪垫，脾，甚至胰腺 11-14 。这些地区的每个人都有自己的优势和挑战。因此，为胰岛移植部位的选择应解决的问题，并在实验的具体情况确定。 </li></ol>据认为，胰岛移植可以克服的局限性的战略，将极大地提高其治疗的潜力。因此，这个协议的一个小鼠模型的详细使用，提供一个有吸引力的方法，确定这种战略。

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<td>Kent Scientific</td>
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<td>Kent Scientific</td>
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<td>USA Scientific</td>
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a method for murine islet isolation and subcapsular kidney transplantation

初以来的巴林杰和Reckard开创性的工作证明，胰岛细胞移植到糖尿病老鼠可以正常化，他们的血糖水平，胰岛移植已提议是一个潜在的治疗1型糖尿病1,2。最近，在人类胰岛移植的进步进一步加强了这种看法 1,3 。但是，两个主要的限制，防止胰岛移植被广泛的临床现实：（一）要求每名病人的胰岛细胞，这严重降低了潜在的接受者的数量大量，及（b）需要沉重的免疫抑制，从而显着影响小儿患者由于其脆弱性，长期免疫抑制人口。策略，可以克服这些限制有可能提高胰岛移植治疗效用。 小鼠肾囊下的胰岛移植是一种被广泛接受的模型，以探讨各种战略，以改善胰岛移植。这个实验需要高品质的小岛和隔离的胰岛细胞植入糖尿病收件人。这两个程序需要可以得到更好的视频演示手术步骤，不是由文字。在这里，我们通过视频和书面协议文件为这些程序的详细步骤。我们还简要地讨论一下不同的移植模型：，同源，同种，同源性自身免疫性，和异体自身免疫性。

Watch this Scientific Journal Video about A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation at JoVE.com

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

分离出胰岛​​移植已被提出，是一个潜在的治疗1型糖尿病。在这里，我们描述的方法，从小鼠胰腺孤立的小岛和移植肾包膜下空间。

小鼠胰岛的分离和包膜下肾移植的方法

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Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

常温<em&gt;离体</em&gt;肾脏的灌注肾的移植保护移植前

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

人类肾脏血管周围间质细胞新型临床分级分离方法

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Orthotopic Rat Kidney Transplantation: A Novel and Simplified Surgical Approach

大鼠原位肾移植: 一种新的简化的手术方法

Kidney transplantation offers increased survival rates and improved quality of life for patients with end-stage renal disease, as compared to any type of ...

Optical Clearing and Imaging of Immunolabeled Kidney Tissue

免疫标记肾组织的光学清除与成像

Optical clearing techniques render tissue transparent by equilibrating the refractive index throughout a sample for subsequent three-dimensional (3-D) ...