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Murine Pancreatic Islets Transplantation under the Kidney Capsule
肾被膜下小鼠胰岛移植   

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参见作者原研究论文

本实验方案简略版
Diabetes
Feb 2006

Abstract

Type 1 diabetes (T1D) is an autoimmune disease caused by the lack of insulin-producing pancreatic beta cells leading to systemic hyperglycemia. Pancreatic islet transplantation is a valid therapeutic approach to restore insulin loss and to promote adequate glycemic control. Pancreatic islet transplantation in mice is an optimal preclinical model to identify new therapeutic strategies aiming at preventing rejection and optimizing post-transplant immuno-suppressive/-tolerogenic therapies.

Islet transplantation in preclinical animal models can be performed in different sites such the kidney capsule, spleen, bone marrow and pancreas. This protocol describes murine islet transplantation under the kidney capsule. This is a widely accepted procedure for research purposes. Stress caused in the animals is minimal and it leads to reliable and reproducible results.

Keywords: Type 1 diabetes (1型糖尿病), Pancreatic islets (胰岛), Islet transplantation (胰岛移植), Murine model (小鼠模型)

Background

Many alternative sites for islet implantation have been reported so far in small animal models and the ideal site must be selected according to the technical advantages of the procedure to be used and for the purpose of the experiments. Bearing in mind that the kidney capsule is an extravascular site and it is not immunoprotected, pancreatic islet transplantation under the kidney capsule remains a surgical procedure with low mortality rates leading to hyperglycemia reversion within a few days. In addition, transplantation under the kidney capsule allows histological studies and formal demonstration of islet function (Cantarelli and Piemonti, 2011; Elisa Cantarelli et al., 2013).

Materials and Reagents

  1. Cotton applicators, sterile (CARLO ERBA Reagents, catalog number: 9.413 161 )
  2. 30 G x ½ in. needle (BD, catalog number: 305106 )
  3. 2 ml slip tip syringe (BD, catalog number: 302204 )
  4. BD INTRAMEDICTM PE 50 (BD, catalog number: B427411 )
  5. Petri dish, Falcon® 50 x 9 mm Sterile (Corning, Falcon®, catalog number: 351006 )
  6. P200 pipette tip (SARSTEDT, catalog number: 70.760.002 )
  7. Eppendorf tubes volume 1.5 ml (Sigma-Aldrich, catalog number: T9661-1000EA)
    Manufacturer: Eppendorf, catalog number: 022363204 .
  8. Silicone tubing adapter (2Biological Instruments, catalog number: SFM3-1550 )
  9. 1 ml syringe with 25 G needle (Ettore Pasquali, catalog number: 11.3500.05 )
  10. Suture Dermalon 5/0 19 MM (Covidien, catalog number: 1756-21 )
  11. Collagenase P (Roche Diagnostics, catalog number: 11213865001 )
  12. Betadine (MEDA PHARMA SpA, Farmacie Coli, catalog number: 023907076 )
  13. Avertin (Sigma-Aldrich, catalog number: T48402 )
  14. Sodium chloride ((NaCl) (CARLO ERBA Reagents, catalog number: FC72101100000 )
  15. Ketoprofen (PFIZER ITALIA Srl DIV.VET)
  16. RPMI 1640 medium (Lonza, catalog number: 12-167F )
  17. L-glutamine (Lonza, catalog number: 17-605E )
  18. HEPES buffer (Lonza, catalog number: 17-737E )
  19. Pen-Strep (Lonza, catalog number: 17-602E )
  20. Fetal bovine serum (Euroclone, catalog number: ECS0180L )
  21. Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14175079 )
  22. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
  23. Histopaque®-1077 (Sigma-Aldrich, catalog number: H8889 )
  24. RPMI/glutamine/HEPES/Pen-Strep/FBS (see Recipes)
  25. HBSS/Ca/HEPES (see Recipes)

Equipment

  1. Forceps (Graefe forceps, 100 mm, curved (ProSciTech, catalog number: T131C ), Tweezers, style 3 (ProSciTech, catalog number: T03-212 )
  2. Scissor (ProSciTech, catalog number: TS103-200SB )
  3. Surgical Shaver (2Biological Instruments, catalog number: 2BTOSRC )
  4. Cautery (Global medical solutions, catalog number: BAA00 )
  5. Thermostatic bath (Thermo Fisher Scientific, Thermo ScientificTM, model: TSGP02 )
  6. Inverted microscope (Compact, Modular Stereo, Leica, model: Leica M60 )
  7. Incubator at 37 °C with 5% CO2, relative humidity ambient to 80% [e.g., Series II 3110 Water-Jacketed CO2 incubators (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM II 3110 Series )]
  8. Pipetman P20/P200/P1000 (Gilson)
  9. Microsyringe 25 μl Hamilton syringe (Hamilton, catalog number: 80401 )
  10. Heating pad 25 x 40 cm, Two Temperature Range (2Biological Instruments, LCPH)
  11. Herasafe KS, Class II biological safety cabinet with UV surface disinfection irradiator (Thermo Fisher Scientific, Thermo ScientificTM, model: HerasafeTM KS II , catalog number: 51022481)

Software

  1. Prism software (GraphPad, USA)

Procedure

  1. Preparation of islets for transplantation
    1. Isolation of pancreatic islet using the appropriate protocol (Graham et al., 2016)
      1. Briefly, sacrifice animal by cervical dislocation, open the mouse abdominal cavity and cut through the peritoneum.
      2. Place 3 ml of collagenase P into a 2 ml slip tip syringe with a 30 G needle. Perform the bile duct injection procedure and dissect the inflated pancreas, being careful not to cut the stomach, the intestines or other abdominal organs.
      3. Pour the inflated pancreases in a collection tube on ice. When all pancreata have been resected, incubate for 15 min at 37 °C in water-bath. Add ice-cold HBSS/Ca/HEPES as quick as possible to stop the digestion process. Disrupt the pancreases by vigorously hand shaking the tubes.
      4. Upon obtaining the digested tissue, the islets are separated by density gradient, recovered from the gradient interface, washed with RPMI 10% FBS and collected in a 50 ml Falcon tube containing 30 ml of RPMI 10% FBS.
        Note: RPMI with serum is demonstrated to maintain (or even augment) glucose-stimulated insulin secretion in murine islets (Andersson, 1978).
    2. Afterwards, 5 ml of islets is transferred into a Petri dish that is gently swirled to collect the islets at the center.
    3. Under an inverted microscope, healthy islets are picked with a P200 sterile pipette tip. Healthy islets have smooth borders in the absence of dark centers (Figure 1). Visual examination of the islets can provide basic information regarding health. Hand-picked healthy islets are then transferred into a new Petri dish containing 3 ml of fresh RPMI 10% FBS.
      Anticipate using one Petri dish for the totality of islets to be injected to one mouse.


      Figure 1. Images of pancreatic islets. Islets isolated before hand-picking (A). Purified islets after hand-picking (B). Scale bars represent 150 µm.

    4. Isolated islets are highly stressed due to physical and chemical processing. To improve overall islet viability, islets cultured in Petri dishes are rested overnight in an incubator at 37 °C with 5% CO2. This allows a better discrimination between dead and alive islets as well as an optimum islets recovery. In case transplantation needs to be performed the same day of islet collection, due to experimental requirements, healthy islets are placed directly into a 1.5 ml Eppendorf tube with 1 ml of sterile RPMI 10% FBS and kept on ice until transplantation. The following day islets are checked under the microscope to confirm viability. If needed, healthy islets are re-collected, leaving behind dead cells. Healthy islets are then transferred from each Petri dish into a sterile 1.5 ml Eppendorf tube containing 1 ml of fresh sterile RPMI 10% FBS (Figure 2).


      Figure 2. Collection and preparation of pancreatic islets. Healthy pancreatic islets transferred into a Petri dish are collected with a P200 (A) and transferred into a sterile 1.5 ml Eppendorf tube (B).

    5. The number of hand-picked islets to be transplanted under the kidney capsule varies from 300 to 600 depending on the experimental conditions required or the mouse strain used (e.g., chemically-induced or spontaneous diabetes, C57BL/6 or NOD, transgenic or knockout) (Battaglia et al., 2006; Gagliani et al., 2011 and 2015; Fousteri et al., 2015a and 2015b) (see Table 1).

      Table 1. Number of pancreatic islets required to reach normoglycemia in different mouse models


  2. Transplantation under the kidney capsule
    1. All instruments and reagents used must be sterile. Ideal transplant recipients should be between 6 to 10 weeks old.
    2. The recipient mouse is anesthetized by an intraperitoneal injection of a weight-adjusted dose of avertin (240 mg/kg).
      Note: Avertin is a quick-acting, non-pharmaceutical grade anesthetic that is used for short duration surgical procedures in mice. It provides rapid and deep anesthesia, followed by fast and full postoperative recovery. This agent is listed on an approved Animal Protocol of the Italian Ministry of Health and procedures are performed by appropriately trained personnel. All guidelines for preparation and storage of avertin are followed.
    3. The level of anesthesia can be tested by pinching the animal’s toes. The mouse’s eyes are kept continuously humid with a physiologic saline solution, 0.9 % NaCl until the mouse awakens to prevent corneal drying.
    4. When anesthesia takes effect, islets are prepared for transplantation. All the islets sedimented (by gravity effect) in the 1.5 ml Eppendorf tube are transferred into a 25 μl Hamilton syringe connected to a P200 tip using the screw mechanism that allows for a slow less strenuous aspiration. Place a silicone tube adapter over the syringe tip. Insert PE50 tubing into the silicone adapter. Turn the Hamilton syringe the opposite site to let the islets into the PE 50 tube. Be careful to not lose the islets from the edge of the tip (Video 1 and Figure 3).


      Figure 3. Collection of pancreatic islets with Hamilton syringe. Pancreatic islets are collected with the Hamilton syringe into a P200 tip (A) and then moved into the PE 50 tube (B).

      Video 1. Preparation of islets for transplantation. Pancreatic islets are collected into a P200 tip and transferred into the PE50 tubing by slowly turned the Hamilton syringe.

    5. While the mouse is under anesthesia, the transplant area is shaved with the electric shears and disinfected with betadine (Video 2).

      Video 2. Mouse preparation for islet transplantation. The transplant area is disinfected and the mouse’s eyes are kept humid.

    6. Localize the kidney using your fingers and make a 1-1.5 cm incision in the skin on the left back side to visualize the peritoneum. Make the 0.5-1 cm incision in the peritoneum to expose the kidney. Slight pressure is applied to both sides of the incision, to allow the kidney to slide out of the abdominal cavity. Keep the surface of the exposed kidney wet with sterile saline using soaked cotton-tipped applicator. Repeat wetting as many times as necessary to prevent the kidney capsule from drying out (Video 3).

      Video 3. Incision and kidney exposure. A small incision is made in the skin and in the peritoneum exposing the kidney.

    7. Make a small scratch on the right flank of the kidney capsule using a syringe 25 gauge needle, to allow the PE50 filled with islets and attached to a Hamilton syringe to be inserted. Reach the posterior end of the capsule and carefully create some space by moving the PE50 tube and slowly inject the islets. Once all islets are injected, carefully remove the PE50 tube, dry the area and cauterize.
      Note: Is highly recommended to use new PE50 tubes for each set of islets to be transplanted to avoid blood clotting. (Video 4 and Figure 4).


      Figure 4. Islets under the kidney capsule. Islets (white) are localized under the kidney capsule. Scale bar represent 3 mm.

      Video 4. Islet transplantation. Pancreatic islets are transplanted under the kidney capsule.

    8. The kidney is then placed back into the cavity and residual blood is cleaned with physiological solution. Both incisions (peritoneum and skin) are sutured with 3 or 4 stitches based on the size of the incision (Video 5 and Figure 5).


      Figure 5. Surgical seams

      Video 5. Closing and mouse revival. The peritoneum and the skin are sutured. Subcutaneous injection of ketoprofen is made and the mice are placed on a heating pad until are active.

    9. The mice are treated with ketoprofen (5 mg/kg) subcutaneously and placed on a heating pad. Animals should be kept warm until they recover from anesthesia and must be monitored until maintaining upright posture and walking normally. Blood glucose levels usually normalize 24 h after the procedure.

Data analysis

  1. In islet transplantation, blood glucose levels are used to define the outcome of islet engraftment, survival, rejection and tolerance according to the transplant model. Islet engraftment is usually defined as the achievement of normoglycemia, while graft rejection is defined by the subsequent development of hyperglycemia. Both in the syngeneic and allogeneic model, islet transplantation failure should be defined either as the inability to reach non-fasting blood glucose levels < 250 mg/dl or death within the first 7 d after islet transplantation (i.e., surgical death). (Cantarelli et al., 2013)
  2. Several drug-based treatments (Gagliani et al., 2011), as well as regulatory cell-based therapies have proved their efficacy in the establishment of long-term tolerance allogeneic murine islet transplantation. To test the development of an active state of tolerance and discard the possibility that treated mice are immune suppressed, mice that do not reject the graft 100 days after transplantation (graft rejection is considered after two consecutive glucose measurements with value > 250 mg/dl) are commonly boosted in vivo with donor-origin splenocytes. A total of 30 x 106 splenocytes isolated from the original islet donor are injected intraperitoneally (i.p.), and blood glucose levels are monitored daily thereafter. Mice still normoglycemic 30-50 days from boosting have developed a long-term tolerance to the allogenic islets (Gagliani et al., 2011).
  3. Statistical analysis
    Islet allograft survival is commonly determined by Kaplan-Meier survival curves and compared using the logrank test. Prism software (GraphPad, USA) is used for all analyses (Fousteri et al., 2015b). When multiple comparisons are made, post hoc comparisons use ANOVA with Bonferroni correction. For two-group comparisons, unpaired two-tailed t-tests with unequal variance are used. In all cases, a two-tailed P value of < 0.05 is considered significant (Battaglia et al., 2006).

Recipes

  1. RPMI/glutamine/HEPES/Pen-Strep/FBS
    500 ml RPMI 1640 (RPMI)
    5 ml L-glutamine
    5 ml HEPES buffer
    5 ml Pen-Strep
    50 ml fetal bovine serum
    Combine all reagents in a sterile biosafety cabinet
    Store at 4 °C
  2. HBSS/Ca/HEPES
    1 L of Hank’s balanced salt solution (HBSS)
    2 mM CaCl2
    20 mM HEPES
    Combine all reagents in a sterile biosafety cabinet
    Store at 4 °C

Acknowledgments

This protocol has been used by members of our laboratory since it was first published (Battaglia et al., 2006) and it was adapted by Gregori and colleagues (Gregori et al., 2015).
The experiments using the mice were performed with approval of and strictly following the guidelines of the Animal Care and Use Committee of the Ospedale San Raffaele and communicated to the Ministry of Health. We would like to thank the members of the group for the support. This protocol is commonly used in our studies of optimizing post-transplant therapeutic strategies in wild-type or transgenic mouse models of type 1 diabetes (Battaglia et al., 2006; Gagliani et al., 2011 and 2015; Fousteri et al., 2015a and 2015b). No potential conflicts of interest were disclosed.

References

  1. Andersson, A. (1978). Isolated mouse pancreatic islets in culture: effects of serum and different culture media on the insulin production of the islets. Diabetologia 14(6): 397-404.
  2. Battaglia, M., Stabilini, A., Draghici, E., Gregori, S., Mocchetti, C., Bonifacio, E. and Roncarolo, M. G. (2006). Rapamycin and interleukin-10 treatment induces T regulatory type 1 cells that mediate antigen-specific transplantation tolerance. Diabetes 55(1): 40-49.
  3. Cantarelli, E., Citro, A., Marzorati, S., Melzi, R., Scavini, M. and Piemonti, L. (2013). Murine animal models for preclinical islet transplantation: No model fits all (research purposes). Islets 5(2): 79-86.
  4. Cantarelli, E. and Piemonti, L. (2011). Alternative transplantation sites for pancreatic islet grafts. Curr Diab Rep 11(5): 364-374.
  5. Fousteri, G., Jofra, T., Di Fonte, R. and Battaglia, M. (2015a). Combination of an antigen-specific therapy and an immunomodulatory treatment to simultaneous block recurrent autoimmunity and alloreactivity in non-obese diabetic mice. PLoS One 10(6): e0127631.
  6. Fousteri, G., Jofra, T., Di Fonte, R., Gagliani, N., Morsiani, C., Stabilini, A. and Battaglia, M. (2015b). Lack of the protein tyrosine phosphatase PTPN22 strengthens transplant tolerance to pancreatic islets in mice. Diabetologia 58(6): 1319-1328.
  7. Gagliani, N., Gregori, S., Jofra, T., Valle, A., Stabilini, A., Rothstein, D. M., Atkinson, M., Roncarolo, M.G., and Battaglia, M. (2011). Rapamycin combined with anti-CD45RB mAb and IL-10 or with G-CSF induces tolerance in a stringent mouse model of islet transplantation. PLoS One 6(12): e28434.
  8. Gagliani, N., Jofra, T., Posgai, A. L., Atkinson, M. A. and Battaglia, M. (2015). Immune depletion in combination with allogeneic islets permanently restores tolerance to self-antigens in diabetic NOD mice. PLoS One 10(11): e0142318.
  9. Graham, K. L., Fynch, S., Papas, E. G., Tan, C., Kay, T. W. and Thomas, H. E. (2016). Isolation and culture of the islets of langerhans from mouse pancreas. Bio-protocol 6(12): e1840.
  10. Gregori S, Mangia P, Bacchetta R, Tresoldi E, Kolbinger F, Traversari C, Carballido JM, de Vries JE, Korthäuer U, Roncarolo MG. (2005). An anti-CD45RO/RB monoclonal antibody modulates T cell responses via induction of apoptosis and generation of regulatory T cells. J Exp Med 201(8): 1293-305.

简介

1型糖尿病(T1D)是由于缺乏产生胰岛素的胰腺β细胞导致系统性高血糖症而引起的自身免疫性疾病。 胰岛移植是恢复胰岛素损失和促进充分血糖控制的有效治疗方法。 小鼠胰岛移植是一种最佳的临床前模型,用于鉴定旨在预防排斥和优化移植后免疫抑制/抗原治疗的新治疗策略。

临床前动物模型中的胰岛移植可以在不同部位进行,如肾囊,脾脏,骨髓和胰腺。 该协议描述了肾囊下的鼠胰岛移植。 这是一个被广泛接受的研究目的。 动物造成的压力很小,并导致可靠和可重复的结果。

【背景】迄今为止在小动物模型中报道了许多用于胰岛植入的备选位置,并且必须根据要使用的程序的技术优势和实验目的来选择理想的位点。 考虑到肾囊是一种血管外部位,并且它没有免疫保护,肾囊下的胰岛移植仍然是一种低死亡率的手术过程,导致几天内高血糖症逆转。 另外,肾囊下的移植允许组织学研究和胰岛功能的正式证明(Cantarelli和Piemonti,2011; Elisa Cantarelli等人,2013)。

关键字:1型糖尿病, 胰岛, 胰岛移植, 小鼠模型

材料和试剂

  1. 棉花涂药器,无菌(CARLO ERBA试剂,目录号:9.413 161)
  2. 30 G x½英寸针(BD,目录号:305106)
  3. 2 ml滑动尖端注射器(BD,目录号:302204)
  4. BD INTRAMEDIC TM PE 50(BD,目录号:B427411)
  5. 培养皿,Falcon 50×9mm无菌培养皿(Corning,Falcon ,产品目录号:351006)
  6. P200枪头(SARSTEDT,目录号:70.760.002)
  7. Eppendorf管体积1.5 ml(Sigma-Aldrich,目录号:T9661-1000EA)
    制造商:Eppendorf,产品目录号:022363204。
  8. 硅胶管适配器(2Biological Instruments,目录号:SFM3-1550)
  9. 1毫升注射器25 G针(Ettore Pasquali,目录号:11.3500.05)
  10. Suture Dermalon 5/0 19 MM(Covidien,目录号:1756-21)
  11. 胶原酶P(Roche Diagnostics,目录号:11213865001)
  12. Betadine(MEDA PHARMA SpA,Farmacie Coli,产品目录号:023907076)
  13. Avertin(Sigma-Aldrich,目录号:T48402)
  14. 氯化钠((NaCl)(CARLO ERBA试剂,目录号:FC72101100000)
  15. 酮洛芬(PFIZER ITALIA Srl DIV.VET)
  16. RPMI 1640培养基(Lonza,目录号:12-167F)
  17. L-谷氨酰胺(Lonza,目录号:17-605E)
  18. HEPES缓冲液(Lonza,目录号:17-737E)
  19. Pen-Strep(Lonza,目录号:17-602E)
  20. 胎牛血清(Euroclone,目录号:ECS0180L)
  21. Hank平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM,目录号:14175079)
  22. 氯化钙二水合物(CaCl 2•2H 2 O)(Sigma-Aldrich,目录号:C5080)
  23. Histopaque-1077(Sigma-Aldrich,目录号:H8889)
  24. RPMI /谷氨酰胺/ HEPES / Pen-Strep / FBS(见食谱)
  25. HBSS / Ca / HEPES(见食谱)

设备

  1. 镊子(Graefe镊子,100毫米,弯曲(ProSciTech,产品目录号:T131C),镊子,样式3(ProSciTech,产品目录号:T03-212)
  2. 剪刀(ProSciTech,产品目录号:TS103-200SB)
  3. 外科剃须刀(2Biological Instruments,目录号:2BTOSRC)
  4. Cautery(全球医疗解决方案,目录号:BAA00)
  5. 恒温浴(Thermo Fisher Scientific,Thermo Scientific TM,型号:TSGP02)
  6. 倒置显微镜(紧凑型,模块化立体声,徕卡,型号:Leica M60)
  7. 培养箱在37℃,5%CO 2,相对湿度环境至80%[例如,Series II 3110 Water-Jacketed CO 2 2培养箱(Thermo Fisher Scientific,Thermo Scientific TM,型号:Forma TM II 3110系列)]
  8. Pipetman P20 / P200 / P1000(吉尔森)
  9. 微量注射器25μl汉密尔顿注射器(汉密尔顿,目录号:80401)
  10. 加热垫25 x 40厘米,两个温度范围(2Biological Instruments,LCPH)
  11. Herasafe KS,具有UV表面消毒辐射器(Thermo Fisher Scientific,Thermo Scientific TM,型号:Herasafe TM KS II,目录号:51022481)的II类生物安全柜。 />

软件

  1. 棱镜软件(GraphPad,美国)

程序

  1. 移植胰岛的制备
    1. 使用适当的方案分离胰岛(Graham et al。,2016)
      1. 简而言之,通过颈椎脱臼牺牲动物,打开小鼠腹腔并切开腹膜。
      2. 将3毫升胶原酶P放入带有30克针的2毫升滑动尖端注射器中。执行胆管注射程序并解剖膨胀的胰腺,注意不要切断胃,肠或其他腹部器官。
      3. 将膨胀的胰脏倒入冰上的收集管中。当所有的胰腺都被切除后,在37℃的水浴中孵育15分钟。尽可能快地加入冰冷的HBSS / Ca / HEPES以停止消化过程。通过剧烈摇动管子来破坏胰脏。
      4. 获得消化的组织后,胰岛通过密度梯度分离,从梯度界面回收,用RPMI 10%FBS洗涤并收集在含有30ml RPMI 10%FBS的50ml Falcon管中。
        注意:含有血清的RPMI在小鼠胰岛中维持(或者甚至增加)葡萄糖刺激的胰岛素分泌(Andersson,1978)。
    2. 之后,将5毫升胰岛转移到培养皿中,轻轻旋转以收集中心的胰岛。
    3. 在倒置显微镜下,用P200无菌吸头吸取健康的胰岛。在没有黑暗中心的情况下,健康的胰岛有光滑的边界(图1)。目测胰岛可以提供有关健康的基本信息。然后将挑选的健康胰岛转移到含有3ml新鲜的RPMI 10%FBS的新培养皿中。
      预计使用一个培养皿将整个小岛注入一只小鼠。

      “”src
      图1.胰岛的图像手工采摘前分离的胰岛(A)。手工采摘后纯化的小岛(B)。比例尺表示150微米。

    4. 由于物理和化学处理,孤立的小岛受到高度压力。为了改善整体胰岛生存力,将在培养皿中培养的胰岛在37℃,5%CO 2的培养箱中静置过夜。这可以更好地区分死亡和活着的胰岛以及最佳的胰岛恢复。如果需要在胰岛采集当天进行移植,由于实验要求,将健康胰岛直接放入装有1ml无菌RPMI 10%FBS的1.5ml Eppendorf管中并保持在冰上直至移植。第二天在显微镜下检查胰岛以确认活力。如果需要的话,重新收集健康的胰岛,留下死细胞。然后将健康的胰岛从每个培养皿转移到含有1ml新鲜无菌RPMI 10%FBS的无菌1.5ml Eppendorf管中(图2)。


      图2.胰岛的收集和制备。用P200(A)收集转移到培养皿中的健康胰岛并转移到无菌1.5ml Eppendorf管(B)中。

    5. 取决于所需的实验条件或使用的小鼠品系(例如,化学诱导的或自发性糖尿病,C57BL / 6),将被移植到肾囊下的手工采集的胰岛的数目从300变化到600或NOD,转基因或敲除)(Battaglia等人,2006; Gagliani等人,2011和2015; Fousteri等人,, 2015a和2015b)(见表1)。

      表1.在不同小鼠模型中达到血糖正常所需的胰岛数目


  2. 在肾囊下移植
    1. 所有使用的仪器和试剂必须是无菌的。理想的移植接受者应该在6至10周之间。
    2. 受体小鼠通过腹膜内注射重量调整剂量的阿佛丁(240mg / kg)麻醉。
      注:Avertin是一种速效非药物级麻醉剂,用于小鼠的短期外科手术。它提供了快速和深度的麻醉,随后快速和完全的术后恢复。该代理被列入意大利卫生部批准的“动物规程”,程序由经过适当培训的人员执行。遵循所有准备和储存阿佛丁的指导原则。
    3. 麻醉水平可以通过捏动物的脚趾来测试。用生理盐水溶液,0.9%NaCl保持小鼠眼睛持续湿润,直到小鼠醒来以防止角膜干燥。
    4. 当麻醉起作用时,胰岛准备移植。在1.5ml Eppendorf管中沉降(通过重力作用)的所有胰岛被转移到25μlHamilton注射器中,该注射器使用螺旋机构连接到P200尖端,从而允许缓慢较少的剧烈抽吸。将硅胶管适配器放在注射器尖端上。将PE50管插入硅胶适配器。将Hamilton注射器转到对面,让胰岛进入PE 50管。小心不要丢失尖端边缘的小岛(视频1和图3)。


      图3.使用汉密尔顿注射器收集胰岛采用Hamilton注射器将胰岛收集到P200吸头(A)中,然后移入PE 50管(B)。

      视频1

    5. 当鼠标处于麻醉状态时,将移植区域用电动剪刀剃光并用betadine消毒(视频2)。

      视频2

    6. 用手指局部放置肾脏,并在左侧背部的皮肤上做一个1-1.5厘米的切口,以观察腹膜。在腹膜上做0.5-1厘米的切口以暴露肾脏。在切口的两侧施加轻微的压力,以允许肾脏滑出腹腔。使用浸泡过的棉签涂抹器保持暴露的肾脏表面用无菌盐水湿润。根据需要重复多次润湿以防止肾囊变干(视频3)。

      视频3

    7. 使用注射器25号针在肾胶囊的右侧面上做小刮擦,以允许插入填充有胰岛并连接到汉密尔顿注射器的PE50。到达胶囊的后端并通过移动PE50管小心地创建一些空间并缓慢注入胰岛。一旦注入了所有的胰岛,请小心地取出PE50管,将该区域烘干并烧灼。
      注意:强烈建议使用新的PE50试管来移植每组胰岛,以避免血液凝结。 (视频4和图4)。

      “”src
      图4.肾囊下的胰岛。 胰岛(白色)位于肾包膜下。比例尺代表3毫米。

      视频4

    8. 然后将肾脏放回空腔中并用生理溶液清洁残余的血液。根据切口的大小(视频5和图5),用3或4针缝合两个切口(腹膜和皮肤)。


      图5.手术缝

      视频5

    9. 用酮洛芬(5mg / kg)皮下处理小鼠并置于加热垫上。动物应保持温暖直至从麻醉中恢复,并且必须进行监测直至保持直立姿势和正常行走。
      24小时后血糖水平通常恢复正常

数据分析

  1. 在胰岛移植中,根据移植模型使用血糖水平来定义胰岛植入,存活,排斥和耐受的结果。胰岛移植通常被定义为实现血糖量正常,而移植排斥则是由高血糖的随后发展定义的。无论在同基因型还是异基因型模型中,胰岛移植失败都应定义为无法达到非空腹血糖水平250毫克/分升或在胰岛移植后7天内死亡(即 ,,手术死亡)。 (Cantarelli et。,2013)
  2. 几项基于药物的治疗(Gagliani等人,2011)以及基于调节性细胞的治疗已证明其在建立长期耐受同种异体小鼠胰岛移植中的功效。为了测试活性状态的耐受性的发展并放弃治疗的小鼠免疫受到抑制的可能性,在移植后100天不排斥移植的小鼠(在两次连续的葡萄糖测量后考虑移植排斥,值> 250mg / dl )通常用供体来源的脾细胞在体内加强。腹膜内(i.p.)注射从原始胰岛供体分离的总共30×10 6脾细胞,然后每日监测血糖水平。
    小鼠血糖升高30-50天仍然具有正常血糖水平,对同种异体胰岛具有长期耐受性(Gagliani et al。 ,2011)。
  3. 统计分析
    通常通过Kaplan-Meier存活曲线确定同种异体胰岛移植物的存活率,并使用logrank测试进行比较。 Prism软件(GraphPad,USA)用于所有分析(Fousteri et。,2015b)。当进行多重比较时,事后比较使用方差分析和Bonferroni校正。对于两组比较,使用不成对的双尾测验。在所有情况下,双尾 P 值< 0.05被认为是显着的(Battaglia等人,2006)。

食谱

  1. RPMI /谷氨酰胺/ HEPES / Pen-Strep / FBS
    500毫升RPMI 1640(RPMI)
    5毫升L-谷氨酰胺
    5毫升HEPES缓冲液
    5毫升Pen-Strep
    50毫升胎牛血清
    将所有试剂组合在无菌生物安全柜中
    在4°C储存
  2. HBSS / Ca / HEPES
    1克Hank平衡盐溶液(HBSS)
    2 mM CaCl 2 2/2 20 mM HEPES
    将所有试剂组合在无菌生物安全柜中
    在4°C储存

致谢

自从该实验室成员首次发表以来,该协议已被使用(Battaglia et al。 2006年),并由Gregori及其同事(Gregori et al。 ,2005)。
使用小鼠的实验经批准并严格按照Ospedale San Raffaele动物护理和使用委员会的指导原则进行并传达给卫生部。我们要感谢小组成员的支持。该方案通常用于优化1型糖尿病的野生型或转基因小鼠模型中的移植后治疗策略的研究(Battaglia等人,2006; Gagliani等人, ,2011年和2015年; Fousteri et al。,2015a和2015b)。没有披露任何潜在的利益冲突。

参考

  1. Andersson,A。(1978)。 培养物中分离的小鼠胰岛:血清和不同培养基对胰岛素产生的影响< / a> Diabetologia 14(6):397-404。
  2. Battaglia,M.,Stabilini,A.,Draghici,E.,Gregori,S.,Mocchetti,C.,Bonifacio,E.和Roncarolo,M.G。(2006)。 雷帕霉素和白细胞介素-10治疗诱导介导抗原特异性移植耐受的T调节1型细胞。 / a> Diabetes 55(1):40-49。
  3. Cantarelli,E.,Citro,A.,Marzorati,S.,Melzi,R.,Scavini,M.和Piemonti,L.(2013)。 临床前胰岛移植的小鼠动物模型:没有模型适合所有人(研究目的)。 胰岛 5(2):79-86。
  4. Cantarelli,E.和Piemonti,L.(2011)。 替代胰岛移植的移植位置 Curr Diab Rep 11(5):364-374。
  5. Fousteri,G.,Jofra,T.,Di Fonte,R.和Battaglia,M.(2015a)。 抗原特异性治疗和免疫调节治疗联合应用可同时阻断非复发性自身免疫和同种异体反应性,肥胖的糖尿病小鼠。 PLoS One 10(6):e0127631。
  6. Fousteri,G.,Jofra,T.,Di Fonte,R.,Gagliani,N.,Morsiani,C.,Stabilini,A。和Battaglia,M.(2015b)。 缺乏蛋白酪氨酸磷酸酶PTPN22可增强移植对小鼠胰岛的耐受性 Diabetologia 58(6):1319-1328。
  7. Gagliani,N.,Gregori,S.,Jofra,T.,Valle,A.,Stabilini,A.,Rothstein,D.M.,Atkinson,M.,Roncarolo,M.G.和Battaglia,M.(2011)。 雷帕霉素与抗CD45RB mAb和IL-10或与G-CSF联合诱导严格的耐受小鼠胰岛移植模型。 PLoS One 6(12):e28434。
  8. Gagliani,N.,Jofra,T.,Posgai,A.L.,Atkinson,M.A。和Battaglia,M。(2015)。 与异体胰岛组合的免疫耗竭可永久恢复糖尿病NOD小鼠对自身抗原的耐受性。 PLoS One 10(11):e0142318。
  9. Graham,K.L.,Fynch,S.,Papas,E.G.,Tan,C.,Kay,T.W。和Thomas,H.E。(2016)。 从小鼠胰腺分离和培养朗格汉斯胰岛 Bio-protocol 6(12):e1840。
  10. Gregori S,Mangia P,Bacchetta R,Tresoldi E,Kolbinger F,Traversari C,Carballido JM,de Vries JE,KorthäuerU,Roncarolo MG。 (2005年)。 抗CD45RO / RB单克隆抗体通过诱导凋亡和产生调节性T细胞。 J Exp Med 201(8):1293-305。
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引用:Jofra, T., Galvani, G., Georgia, F., Silvia, G., Gagliani, N. and Battaglia, M. (2018). Murine Pancreatic Islets Transplantation under the Kidney Capsule. Bio-protocol 8(5): e2743. DOI: 10.21769/BioProtoc.2743.
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