Name: inguinal subcutaneous white adipose tissue (iswat) transplantation model of murine islets
Uploaded: 2020-02-16T15:00:00
Description: Watch this Scientific Journal Video about Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets at JoVE.com

这项工作得到了国家重点研发项目（2017YFC1103704）广东省高水平医院建设专项资金（2019年）、深圳三明医药项目（SZSM201412020）、高水平基金的资助。深圳市医学学科建设（2016031638），深圳市科技基金会（JCJY20160260229204849975，GJHZ2017031417171357556），深圳市卫生和计划生育基金会（SZXJ2017021SZXJ2018059），医疗中国广东省科学研究基金会（A2019218），中国博士后科学基金会（2018M633218）。

本议定书的所有程序均遵循深圳市第二人民医院伦理审查委员会的动物福利原则。从广东省医学动物中心购买的8至10周大的C57BL/6雄性小鼠为岛移植受体和捐赠者。收获细胞的收获、隔离、培养或管理程序是在无菌条件下进行的。
1. 岛准备
<ol><li>准备胶原酶 V 型工作溶液。称量胶原酶V型，用D-Hank的缓冲液溶解至1mg/mL的最终浓度，使用0.22μm注射器驱动的过滤单元和60 mL注射器?...

<ol>
	<li>Cho, N. 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=IDF+Diabetes+Atlas:+Global+estimates+of+diabetes+prevalence+for+2017+and+projections+for+2045.">IDF Diabetes Atlas: Global estimates of diabetes prevalence for 2017 and projections for 2045.</a> Diabetes Research and Clinical Practice. 138, 271-281 (2018).</li><li>Shapiro, A. M., 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=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> New England Journal of Medicine. 343 (4), 230-238 (2000).</li><li>McCall, M., 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=Update+on+islet+transplantation.">Update on islet transplantation.</a> Cold Spring Harbor Perspectives in Medicine. 2 (7), 007823 (2012).</li><li>Pathak, V., Pathak, N. M., O'Neill, C. L., Guduric-Fuchs, J., Medina, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapies+for+Type+1+Diabetes:+Current+Scenario+and+Future+Perspectives.">Therapies for Type 1 Diabetes: Current Scenario and Future Perspectives.</a> Clinical Medicine Insights: Endocrinology and Diabetes. 12, 1179551419844521 (2019).</li><li>Bottino, R., Knoll, M. F., Knoll, C. A., Bertera, S., Trucco, M. 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+Future+of+Islet+Transplantation+Is+Now.">The Future of Islet Transplantation Is Now.</a> Frontiers in Medicine (Lausanne). 5, 202 (2018).</li><li>Stokes, R. A., 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=Transplantation+sites+for+human+and+murine+islets.">Transplantation sites for human and murine islets.</a> Diabetologia. 60 (10), 1961-1971 (2017).</li><li>van der Windt, D. J., Echeverri, G. J., Ijzermans, J. N., Cooper, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+choice+of+anatomical+site+for+islet+transplantation.">The choice of anatomical site for islet transplantation.</a> Cell Transplantation. 17 (9), 1005-1014 (2008).</li><li>Addison, P., Fatakhova, K., Rodriguez Rilo, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+for+an+Alternative+Site+of+Islet+Cell+Transplantation.">Considerations for an Alternative Site of Islet Cell Transplantation.</a> Journal of Diabetes Science and Technology. , (2019).</li><li>Pepper, A. R., Bruni, A., 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=Clinical+islet+transplantation:+is+the+future+finally+now.">Clinical islet transplantation: is the future finally now.</a> Current Opinion in Organ Transplantation. 23 (4), 428-439 (2018).</li><li>Bellin, M. D., 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=Similar+islet+function+in+islet+allotransplant+and+autotransplant+recipients,+despite+lower+islet+mass+in+autotransplants.">Similar islet function in islet allotransplant and autotransplant recipients, despite lower islet mass in autotransplants.</a> Transplantation. 91 (3), 367-372 (2011).</li><li>Bruni, A., Gala-Lopez, B., Pepper, A. R., Abualhassan, N. S., Shapiro, A. J. <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+for+the+treatment+of+type+1+diabetes:+recent+advances+and+future+challenges.">Islet cell transplantation for the treatment of type 1 diabetes: recent advances and future challenges.</a> Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy. 7, 211-223 (2014).</li><li>Smood, B., Bottino, R., Hara, H., Cooper, D. K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+renal+subcapsular+space+the+preferred+site+for+clinical+porcine+islet+xenotransplantation?+Review+article.">Is the renal subcapsular space the preferred site for clinical porcine islet xenotransplantation? Review article.</a> International Journal of Surgery and Medicine. 69, 100-107 (2019).</li><li>Luan, N. M., Iwata, H. <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+allogeneic+islet+graft+survival+in+prevascularized+subcutaneous+sites+without+immunosuppressive+treatment.">Long-term allogeneic islet graft survival in prevascularized subcutaneous sites without immunosuppressive treatment.</a> American Journal of Transplantation. 14 (7), 1533-1542 (2014).</li><li>Yasunami, Y., 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+Novel+Subcutaneous+Site+of+Islet+Transplantation+Superior+to+the+Liver.">A Novel Subcutaneous Site of Islet Transplantation Superior to the Liver.</a> Transplantation. 102 (6), 945-952 (2018).</li><li>Rajab, 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:+alternative+sites.">Islet transplantation: alternative sites.</a> Current Diabetes Reports. 10 (5), 332-337 (2010).</li><li>Ekser, B., Vagefi, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Search+for+the+best+site+in+islet+xenotransplantation.">Search for the best site in islet xenotransplantation.</a> International Journal of Surgery and Medicine. 70, 106-107 (2019).</li><li>Lu, Y., 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+Method+for+Islet+Transplantation+to+the+Omentum+in+Mouse.">A Method for Islet Transplantation to the Omentum in Mouse.</a> Journal of Visualized Experiments. (143), e57160 (2019).</li><li>Neuman, J. C., Truchan, N. A., Joseph, J. W., Kimple, M. E. <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+mouse+pancreatic+islet+isolation+and+intracellular+cAMP+determination.">A method for mouse pancreatic islet isolation and intracellular cAMP determination.</a> Journal of Visualized Experiments. (88), e50374 (2014).</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> Journal of Visualized Experiments. (50), e2096 (2011).</li><li>Khatri, R., Hussmann, B., Rawat, D., Gurol, A. O., Linn, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraportal+Transplantation+of+Pancreatic+Islets+in+Mouse+Model.">Intraportal Transplantation of Pancreatic Islets in Mouse Model.</a> Journal of Visualized Experiments. (135), e57559 (2018).</li><li>Carter, J. D., Dula, S. B., Corbin, K. L., Wu, R., Nunemaker, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+rodent+islet+isolation+and+assessment.">A practical guide to rodent islet isolation and assessment.</a> Biological Procedures Online. 11, 3-31 (2009).</li></ol>

胰腺胰岛移植是治疗T1DM的一种有前途的治疗方法。这种疗法的效果受多种因素的影响，选择最佳的植入部位是非常重要的。胰岛移植的理想解剖部位应具有以下特征：简单移植、活检和移植检索程序的可及性;减少并发症;血糖控制成功率高;和长期存活的小岛移植15，16。
我们的团队之前描述了在鼠模17的阴岛移植?...

根据国际糖尿病联合会（IDF）1的统计数据，全球1型糖尿病的发病率和患病率正在迅速上升。岛移植是治疗T1DM4最有希望的方法之一。自从使用埃德蒙顿协议2的临床小岛移植取得重大突破以来，5年后T1DM受体的功能性小岛移植存活率现已达到50%左右。
过去，在实验岛移植5、6、7等移植部位，如肝、肾胶囊、脾脏、肌肉内区域、皮下空间、骨髓、眼袋等，上述一些站点已在临床环境中测试8。虽然小岛移植到肝脏仍然是目前临床应用最广泛的方法，但在使用这个部位时，有几个重要问题需要解决。例如，如何减少由即时血液介导炎症反应（IBMIR）和不良氧合供应10，11引起的移植胰岛的早期损失，以及如何在必要时取回胰岛移植物，因为它们在肝脏中分散局部化。肾脏胶囊可能是啮齿动物接受者的理想部位。然而，紧肾胶囊限制了移植足够的异体小岛在人类，虽然它可能是一个更好的适合胰岛异种移植由于高度纯化的猪胰岛制剂临床使用5，12。因此，寻找更合适的岛移植部位正在进行中。
皮下空间可作为临床上适用于小岛移植的场所，因为它可及性。然而，胰岛移植到皮下空间的效率极低，因此需要相对大量的胰岛来逆转高血糖13。最近，日本一个研究小组发现ISWAT，一种与肝脏14相比，在小鼠模型中用于小岛移植的新型皮下部位。ISWAT包含外胃动脉和静脉，因此丰富的血液供应可以保证小岛移植再血管化。在这份手稿中，我们提出了一种简单的植入方法，使用基底膜基质水凝胶来修复ISWAT中的合成鼠岛。该协议证明对小岛移植有效。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#956;m Syringe-driven Filter Unit</td>
			<td>Merck Millipore</td>
			<td>SLHV033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL centrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Pasteur pipette</td>
			<td>JingAn Biological, China</td>
			<td>J00085</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>Szboon, China</td>
			<td>20170829</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 surgical suture</td>
			<td>sh-Jinhuan, China</td>
			<td>CR537</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mL syringe</td>
			<td>Szboon, China</td>
			<td>20170623</td>
			<td></td>
		</tr>
		<tr>
			<td>75% Ethanol</td>
			<td>LIRCON, China</td>
			<td>9180527</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-mouse IgG(H+L)</td>
			<td>Invitrogen</td>
			<td>A21202</td>
			<td>Dilution (1:200)</td>
		</tr>
		<tr>
			<td>anti-mouse Glucagon antibody</td>
			<td>Abcam</td>
			<td>ab10988</td>
			<td>Dilution (1:100)</td>
		</tr>
		<tr>
			<td>anti-mouse insulin antibody</td>
			<td>Cell Signaling Technology</td>
			<td>3014s</td>
			<td>Dilution (1:100)</td>
		</tr>
		<tr>
			<td>blunt-pointed perfusion needle</td>
			<td>Oloey, China</td>
			<td>005</td>
			<td>32G, yellow</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Meilune, China</td>
			<td>MB4219</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 Mice</td>
			<td>Medical Animal Center of Guangdong Province</td>
			<td></td>
			<td>8~10 weeks</td>
		</tr>
		<tr>
			<td>cell culture dish</td>
			<td>BIOFIL, China</td>
			<td>TCD000100</td>
			<td>General,Non-treated,87.8 mm diameter</td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Thermo Scientific</td>
			<td>ST16R</td>
			<td></td>
		</tr>
		<tr>
			<td>cephalosporin</td>
			<td>Lukang medical, China</td>
			<td>150303</td>
			<td></td>
		</tr>
		<tr>
			<td>CMRL-1066</td>
			<td>Sigma-Aldrich</td>
			<td>C0422</td>
			<td></td>
		</tr>
		<tr>
			<td>Codos Pet Clipper</td>
			<td>Szcodos, China</td>
			<td>CP-8000</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase Type V</td>
			<td>Sigma</td>
			<td>C9262</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fisher</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>D-hank&#39;s buffer</td>
			<td>Coolaber, China</td>
			<td>PM5140-10</td>
			<td></td>
		</tr>
		<tr>
			<td>dithizone</td>
			<td>Sigma-Aldrich</td>
			<td>D5130</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I</td>
			<td>Sigma-Aldrich</td>
			<td>D4263</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin staining media</td>
			<td>Beyotime Biotech, China</td>
			<td>C0109</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30084</td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescein diacetate (FDA)</td>
			<td>Thermo Fisher</td>
			<td>F1303</td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescent microscope</td>
			<td>Leica</td>
			<td>DMIL</td>
			<td></td>
		</tr>
		<tr>
			<td>gel-loading pipet tips</td>
			<td>Corning</td>
			<td>CLS4884</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Coolaber, China</td>
			<td>PM5150-10</td>
			<td></td>
		</tr>
		<tr>
			<td>hematoxylin staining media</td>
			<td>Cell Signaling Technology</td>
			<td>14166S</td>
			<td></td>
		</tr>
		<tr>
			<td>HISTOPAQUE-1077</td>
			<td>Sigma-Aldrich</td>
			<td>RNBG0522</td>
			<td></td>
		</tr>
		<tr>
			<td>HISTOPAQUE-1119</td>
			<td>Sigma-Aldrich</td>
			<td>RNBG0536</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogel</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>Basement Membrane Matrix</td>
		</tr>
		<tr>
			<td>Iodophor</td>
			<td>LIRCON, China</td>
			<td>5190313</td>
			<td></td>
		</tr>
		<tr>
			<td>light-tight culture dish</td>
			<td>DVS, China</td>
			<td>AN-5058548</td>
			<td>self-made, glass dish sprayed with black paint</td>
		</tr>
		<tr>
			<td>Medical Adhesive Tape</td>
			<td>Cofoe, China</td>
			<td>K12001</td>
			<td></td>
		</tr>
		<tr>
			<td>non-invasive microtweezers</td>
			<td>RWD Life Science</td>
			<td>F11033-11 and F12016-15</td>
			<td></td>
		</tr>
		<tr>
			<td>One Touch ultraeasy Basic blood glucose monitoring system</td>
			<td>Johnson &#38; Johnson</td>
			<td>33391713</td>
			<td></td>
		</tr>
		<tr>
			<td>ophthalmic scissors</td>
			<td>RWD Life Science</td>
			<td>S12012-12 and S11001-08</td>
			<td></td>
		</tr>
		<tr>
			<td>P/S (penicillin / streptomycin)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>pentobarbital sodium</td>
			<td>Sigma-Aldrich</td>
			<td>P-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma-Aldrich</td>
			<td>P4864</td>
			<td></td>
		</tr>
		<tr>
			<td>STZ (streptozotocin)</td>
			<td>Sigma-Aldrich</td>
			<td>S0130</td>
			<td></td>
		</tr>
		<tr>
			<td>Test Strip</td>
			<td>GenUltimate</td>
			<td>100-50</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITC-conjugated Goat anti-Rabbit IgG(H+L)</td>
			<td>proteintech</td>
			<td>SA00007-2</td>
			<td>Dilution (1:200)</td>
		</tr>
		<tr>
			<td>vascular clamp</td>
			<td>RWD Life Science</td>
			<td>R31006-04</td>
			<td></td>
		</tr>
	</tbody>
</table>

inguinal subcutaneous white adipose tissue (iswat) transplantation model of murine islets

胰腺胰岛移植是治疗1型糖尿病的一种成熟疗法。肾胶囊是啮齿动物模型中最常见的岛移植部位。然而，紧密的肾胶囊限制了大型动物和人类足够的胰岛移植。阴皮下皮白脂肪组织（ISWAT）是一种新的皮下空间，被发现是小岛移植的潜在价值部位。该网站有比其他皮下空间更好的血液供应。此外，ISWAT容纳比肾胶囊更大的小岛质量，移植到它很简单。本手稿描述了在ISWAT的协同糖尿病小鼠受体的小鼠小岛分离和移植程序。使用该协议，通过标准胶原酶消化分离了鼠胰腺胰岛，并用基底膜基质水凝胶固定ISWAT部位的纯化胰岛。对受体小鼠的血糖水平进行了100多天的监测。在移植后的第100天检索了岛移植物，进行组织学分析。本手稿中描述的 ISWAT 站点中的岛移植方案简单而有效。

该协议引入了两个程序：鼠小岛制备和滑入ISWAT部位的岛移植。在第一个过程中，在用V型胶原酶溶液渗透和消化后，用Histopaque-1119和Histopaque-1077和额外的手工拾取步骤进行纯化，分离的鼠岛将足够纯，用于移植（如图1所示），具有高存活率的分离胰岛将用于移植（如图2所示）。在第二个程序中，使用STZ化学的糖尿病诱导至关重要。STZ的最佳剂量取决?...

Watch this Scientific Journal Video about Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets at JoVE.com

Inguinal Subcutaneous White Adipose Tissue ISWAT Transplantation Model of Murine Islets

在本协议中，描述了一种将鼠小岛分离和移植到皮下白脂肪组织的方法。分离的银体鼠胰岛使用基底膜水凝胶移植到鼠细胞接受者中。监测受体的血糖水平，对小岛移植物进行组织学分析。

毛里岛皮下白脂肪组织（ISWAT）移植模型

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for ...

岛移植是治疗一型糖尿病最有前途的方法之一。目前正在寻找合适的小岛移植地点。对皮下脂肪组织的岛体移植是一种简单而有效的方法，可能具有临床意义。将这个程序推断到诊所可能导致一种治疗一型糖尿病患者的治疗方法的发展。这种方法可以提供临床小岛移植的见解，因为语言已被确定不是一个理想的地点，为小岛移植。必须仔细遵循程序的每一步，特别是关键步骤，如胰腺消化。视觉演示将帮助研究人员对这项技术进行新的研究，以更好地确定如何执行小岛隔离和转换。对于小岛收获，首先用75%乙醇喷洒10周老的C57黑色六鼠的皮毛，然后用新鲜准备的胶原酶溶液填充5毫升注射器。用 32 量弯曲钝尖灌注针为注射器，将鼠标放在冰袋上的超活位置。使用直尖眼科剪刀在阴区皮肤上做一个横向开口，并向动物头部伸出切口。通过 V 切口完全打开腹部，从阴区到西腓体过程，并移动肝脏暴露胆囊和普通胆管的整个长度。然后使用血管夹关闭普通胆管的十二指肠开口，并在胆囊中切开一个小开口。用先前准备的灌注针将普通胆汁导管通过开口，将大约两毫升胶原酶溶液注射到胰腺中。当所有的胶原蛋白酶已经交付，使用两对非侵入性微型钳子，将充满的胰腺从肠道、胃和脾脏分离，并将胰腺放入50毫升的圆锥形管中。收集完所有样本后，每胰腺加入100微升Dnase I，并在10秒内用力摇动每根管子约40次，然后将组织放入37摄氏度的水浴中，进行3至5分钟的中度摇晃。在孵育结束时，加起来50毫升的停止溶液，以停止消化的最终体积。脉冲离心管在4摄氏度时以750倍G的速度离心，然后迅速停止离心机。丢弃超钠后，在相同离心条件下，每次洗涤的止损溶液为 15 至 25 毫升，洗涤两次。第二次洗涤后，将每管三粒颗粒放入新的50毫升锥形管中。并重新暂停胰腺细胞在总体积为10毫升的Stopaque1119。接下来，在每个管的侧面小心地添加五毫升的 Histopaque 1077，然后是五毫升的 HBSS。通过离心分离细胞，并使用五毫升巴斯德移液器从HSSS Histopaque 1077接口收集小岛。将小岛汇集到新的 50 毫升锥形管中，最终体积为 50 毫升停止溶液，用于脉冲离心，并丢弃上流水线。将颗粒重新在 30 毫升新鲜停止溶液中，进行一分钟的离心，并在 30 毫升培养培养的培养中重新供应小岛。将小岛放入每管一个 10 厘米的光紧培养盘中。把盘子放在立体显微镜下。使用 200 微升凝胶加载移液器尖端，从溶液中手动挑选白色球形小岛。将小岛放入未经处理的细胞培养皿中，在收获时含有10毫升培养培养剂。在光学显微镜下通过二染色检查手工采摘的小岛纯度。并在荧光显微镜下通过荧光素二甲酸酯丙酸丙酸酯染色来检测小岛的可行性。然后在95%的空气和5%的二氧化碳中培养新鲜培养基的孤立小岛，直到移植。从糖尿病诱导后的第三天和第四天开始，穿刺每个链球菌素注射8周男性C57黑色六只小鼠的骨静脉.m大约10：00左右，从每个接受动物采集血液样本。使用基本的血糖监测仪器确定每只小鼠的不禁食血糖水平，然后将血液添加到单独的测试条上。当动物连续两天表现出每升至少20毫摩尔的血糖水平时，称重并标记受助小鼠，并确认麻醉动物对踏板反射缺乏反应。将零下20摄氏度的西气凝胶储存在冰上进行解冻。将每个接受者450至500个小岛当量转移到无菌的1.5毫升离心管中，每管含有200微升的介质。用75%乙醇擦拭接受者的左内，将鼠标放在上角位置。用手术胶带固定四肢，并使用剪子去除手术地点周围的毛发。用碘磷擦拭手术区域，在消毒的皮肤中进行垂直切口。识别 ISWAT 内的劣质胃动脉和静脉，并在容器上方创建一个小口袋。通过离心沉淀小岛，并尽可能去除上流液。在每个管中加入20微升解冻水凝胶，轻轻重新暂停小岛，注意避免气泡。获得均匀悬浮液后，使用 200 微升移液器尖端小心地将整个小岛水凝胶混合物从一根管中放入口袋。当水凝胶完全凝固后，在移植现场加入20微升头孢菌素，用5-0连续手术缝合关闭肌肉和皮肤。然后将接收者放入热垫上的清洁笼中，进行监控，直至完全恢复。如证明，经过穆林岛处理后，分离的穆林岛将足够纯正，适合移植。只有具有高生存能力的孤立小岛才应用于移植。将小岛嫁接物与水凝胶彻底混合后，可将嫁接物植入 ISWAT 站点。在移植到 ISWAT 约一个月后，小岛移植逆转高血糖，同时接受小鼠的体重逐渐增加。移植后100天，移植物的切除导致10：00的10：00.m。非禁食血糖水平。组织学分析此时点，发现小岛嫁接一直完好无损。胰岛素和胰高血糖系统免疫荧光染色表明，移植的胰岛在移植后100天也发挥作用。小心不要太有力地摇动蛋白胰腺细胞，否则获得的胰岛的能力将受到损害。分离的小岛可以在肾胶囊下移植作为控制，以比较分离移植到地位下皮下白脂肪组织中的影响。该程序将增加小岛移植的临床试验的开始到除肝脏以外的其他地点。

Research

JoVE Journal

Medicine

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

自动子宫组织移植手术诱发子宫内膜异位症小鼠模型

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of ...

科研

医学

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

闭塞性细支气管炎在原位肺移植小鼠模型的发展

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

用改进的袖套法小鼠颈部心脏移植模型

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

肺动脉瓣使用异位心脏移植小鼠模型移植

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

小鼠角膜移植模型，研究实体器官移植的最常见形式

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

封装产热前脂肪细胞移植到脂肪组织仓库

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

皮层下的多功能小鼠模型白质行程轴索变性和白质神经生物学的研究

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

创作和移植的糖尿病伤口愈合模型中脂肪源性干细胞 (ASC) 表

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

经非透法皮下腹脂肪组织活检技术

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

脂肪组织干细胞移植对大鼠部分结肠切除术后渗漏的减少

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...