毛里岛皮下白脂肪组织（ISWAT）移植模型
1Shenzhen Xenotransplantation Medical Engineering Research and Development Center, Institute of Translational Medicine, Shenzhen Second People’s Hospital,First Affiliated Hospital of Shenzhen University, Health Science Center, 2Institute for Cellular Therapeutics,Allegheny-Singer Research Institute, 3School of Pharmaceutical Sciences,Wenzhou Medical University, 4Xenotransplantation Program/Department of Surgery,University of Alabama at Birmingham, 5Jiangsu Key Laboratory of Xenotransplantation,Nanjing Medical University
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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?…
Representative Results
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?…
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| 0.22 μm Syringe-driven Filter Unit | Merck Millipore | SLHV033RB | |
| 1.5 mL centrifuge tube | Axygen | MCT-150-C | |
| 5 mL Pasteur pipette | JingAn Biological, China | J00085 | |
| 5 mL syringe | Szboon, China | 20170829 | |
| 50 mL conical tube | Corning | 430829 | |
| 5-0 surgical suture | sh-Jinhuan, China | CR537 | |
| 60 mL syringe | Szboon, China | 20170623 | |
| 75% Ethanol | LIRCON, China | 9180527 | |
| Alexa Fluor 488 donkey anti-mouse IgG(H+L) | Invitrogen | A21202 | Dilution (1:200) |
| anti-mouse Glucagon antibody | Abcam | ab10988 | Dilution (1:100) |
| anti-mouse insulin antibody | Cell Signaling Technology | 3014s | Dilution (1:100) |
| blunt-pointed perfusion needle | Oloey, China | 005 | 32G, yellow |
| BSA | Meilune, China | MB4219 | |
| C57BL/6 Mice | Medical Animal Center of Guangdong Province | 8~10 weeks | |
| cell culture dish | BIOFIL, China | TCD000100 | General,Non-treated,87.8 mm diameter |
| centrifuge | Thermo Scientific | ST16R | |
| cephalosporin | Lukang medical, China | 150303 | |
| CMRL-1066 | Sigma-Aldrich | C0422 | |
| Codos Pet Clipper | Szcodos, China | CP-8000 | |
| collagenase Type V | Sigma | C9262 | |
| DAPI | Thermo Fisher | D1306 | |
| D-hank's buffer | Coolaber, China | PM5140-10 | |
| dithizone | Sigma-Aldrich | D5130 | |
| Dnase I | Sigma-Aldrich | D4263 | |
| Eosin staining media | Beyotime Biotech, China | C0109 | |
| FBS | GE Healthcare Life Sciences | SH30084 | |
| fluorescein diacetate (FDA) | Thermo Fisher | F1303 | |
| fluorescent microscope | Leica | DMIL | |
| gel-loading pipet tips | Corning | CLS4884 | |
| HBSS | Coolaber, China | PM5150-10 | |
| hematoxylin staining media | Cell Signaling Technology | 14166S | |
| HISTOPAQUE-1077 | Sigma-Aldrich | RNBG0522 | |
| HISTOPAQUE-1119 | Sigma-Aldrich | RNBG0536 | |
| Hydrogel | BD Biosciences | 356234 | Basement Membrane Matrix |
| Iodophor | LIRCON, China | 5190313 | |
| light-tight culture dish | DVS, China | AN-5058548 | self-made, glass dish sprayed with black paint |
| Medical Adhesive Tape | Cofoe, China | K12001 | |
| non-invasive microtweezers | RWD Life Science | F11033-11 and F12016-15 | |
| One Touch ultraeasy Basic blood glucose monitoring system | Johnson & Johnson | 33391713 | |
| ophthalmic scissors | RWD Life Science | S12012-12 and S11001-08 | |
| P/S (penicillin / streptomycin) | Gibco | 15140-122 | |
| pentobarbital sodium | Sigma-Aldrich | P-010 | |
| Propidium iodide | Sigma-Aldrich | P4864 | |
| STZ (streptozotocin) | Sigma-Aldrich | S0130 | |
| Test Strip | GenUltimate | 100-50 | |
| TRITC-conjugated Goat anti-Rabbit IgG(H+L) | proteintech | SA00007-2 | Dilution (1:200) |
| vascular clamp | RWD Life Science | R31006-04 |
Referências
- Cho, N. H., et al. IDF Diabetes Atlas: Global estimates of diabetes prevalence for 2017 and projections for 2045. Diabetes Research and Clinical Practice. 138, 271-281 (2018).
- Shapiro, A. M., et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. New England Journal of Medicine. 343 (4), 230-238 (2000).
- McCall, M., Shapiro, A. M. Update on islet transplantation. Cold Spring Harbor Perspectives in Medicine. 2 (7), 007823 (2012).
- Pathak, V., Pathak, N. M., O’Neill, C. L., Guduric-Fuchs, J., Medina, R. J. Therapies for Type 1 Diabetes: Current Scenario and Future Perspectives. Clinical Medicine Insights: Endocrinology and Diabetes. 12, 1179551419844521 (2019).
- Bottino, R., Knoll, M. F., Knoll, C. A., Bertera, S., Trucco, M. M. The Future of Islet Transplantation Is Now. Frontiers in Medicine (Lausanne). 5, 202 (2018).
- Stokes, R. A., et al. Transplantation sites for human and murine islets. Diabetologia. 60 (10), 1961-1971 (2017).
- van der Windt, D. J., Echeverri, G. J., Ijzermans, J. N., Cooper, D. K. The choice of anatomical site for islet transplantation. Cell Transplantation. 17 (9), 1005-1014 (2008).
- Addison, P., Fatakhova, K., Rodriguez Rilo, H. L. Considerations for an Alternative Site of Islet Cell Transplantation. Journal of Diabetes Science and Technology. , (2019).
- Pepper, A. R., Bruni, A., Shapiro, A. M. J. Clinical islet transplantation: is the future finally now. Current Opinion in Organ Transplantation. 23 (4), 428-439 (2018).
- Bellin, M. D., et al. Similar islet function in islet allotransplant and autotransplant recipients, despite lower islet mass in autotransplants. Transplantation. 91 (3), 367-372 (2011).
- Bruni, A., Gala-Lopez, B., Pepper, A. R., Abualhassan, N. S., Shapiro, A. J. Islet cell transplantation for the treatment of type 1 diabetes: recent advances and future challenges. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy. 7, 211-223 (2014).
- Smood, B., Bottino, R., Hara, H., Cooper, D. K. C. Is the renal subcapsular space the preferred site for clinical porcine islet xenotransplantation? Review article. International Journal of Surgery and Medicine. 69, 100-107 (2019).
- Luan, N. M., Iwata, H. Long-term allogeneic islet graft survival in prevascularized subcutaneous sites without immunosuppressive treatment. American Journal of Transplantation. 14 (7), 1533-1542 (2014).
- Yasunami, Y., et al. A Novel Subcutaneous Site of Islet Transplantation Superior to the Liver. Transplantation. 102 (6), 945-952 (2018).
- Rajab, A. Islet transplantation: alternative sites. Current Diabetes Reports. 10 (5), 332-337 (2010).
- Ekser, B., Vagefi, P. A. Search for the best site in islet xenotransplantation. International Journal of Surgery and Medicine. 70, 106-107 (2019).
- Lu, Y., et al. A Method for Islet Transplantation to the Omentum in Mouse. Journal of Visualized Experiments. (143), e57160 (2019).
- Neuman, J. C., Truchan, N. A., Joseph, J. W., Kimple, M. E. A method for mouse pancreatic islet isolation and intracellular cAMP determination. Journal of Visualized Experiments. (88), e50374 (2014).
- Zmuda, E. J., Powell, C. A., Hai, T. A method for murine islet isolation and subcapsular kidney transplantation. Journal of Visualized Experiments. (50), e2096 (2011).
- Khatri, R., Hussmann, B., Rawat, D., Gurol, A. O., Linn, T. Intraportal Transplantation of Pancreatic Islets in Mouse Model. Journal of Visualized Experiments. (135), e57559 (2018).
- Carter, J. D., Dula, S. B., Corbin, K. L., Wu, R., Nunemaker, C. S. A practical guide to rodent islet isolation and assessment. Biological Procedures Online. 11, 3-31 (2009).
Citar este artigo
Peng, Y., Zou, Z., Chen, J., Zhang, H., Lu, Y., Bittino, R., Fu, H., Cooper, D. K. C., Lin, S., Cao, M., Dai, Y., Cai, Z., Mou, L. Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets. J. Vis. Exp. (156), e60679, doi:10.3791/60679 (2020).