补肾胶囊对糖尿病小鼠的胰岛移植

Published 10/31/2007
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Summary

我们的协议是干净,便捷地提供胰岛细胞或细胞小鼠肾囊下。集中使用,用于移植的肾囊下细胞最终油管颗粒细胞。这项技术的易用性,减少压力的细胞和鼠标。

Cite this Article

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Szot, G. L., Koudria, P., Bluestone, J. A. Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice. J. Vis. Exp. (9), e404, doi:10.3791/404 (2007).

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Abstract

我们的协议是干净,便捷地提供胰岛细胞或细胞,糖尿病患者或正常小鼠肾囊下。我们发现,这是更容易集中到最终交付油管,用于移植的肾囊下细胞(PE50)颗粒的胰岛细胞或细胞。这种技术提供的速度和易用性,同时减少任何不必要的压力细胞或鼠标。

载入:落户,手工采摘,胰岛细胞或颗粒细胞仔细吸气使用P200 pipetteman和一条直线,薄壁枪头的1.5 ml离心管底部。 PE50管材的长度是使用一个小的硅胶适配器管连接到枪头。细胞是可以解决,在一角,然后慢慢拨号pipetteman PE50管材。一旦细胞附近PE50管材年底,扭结和硅胶适配器管放置在扭结。 PE50管材被转移到一个15毫升锥形含有削减5毫升吸管,PE50管材超过5毫升吸管一边录音,以防止在离心冰壶。细胞可以达到1000转和停止。

移植受体小鼠麻醉,剃光,并清理。一个小切口鼠标左翼和肾脏暴露出来。肾,脂肪和组织保持与生理盐水棉签湿润。使用硅适配器管,是连接到远端的PE50汉密尔顿螺杆驱动注射器,包含一个枪头,。一个小缺口是对肾脏的右侧边,不是太大,也不太深。 PE50管材的斜面结束,最近的细胞,是经过精心包膜下,油管是移动轻轻地擦拭时生理盐水以腾出空间;干胶囊可以容易撕裂。一个小气泡慢慢拨号注射器螺杆驱动包膜下交付。胰岛细胞,然后慢慢地交付背后的气泡。一旦胰岛已交付的肾动态平衡是维持和装饰品是低热量烧灼。肾脏是放回腔和腹膜和皮肤缝合装订。老鼠立即治疗氟尼辛和丁丙诺啡平方米,并放置在笼子里加热垫。

Protocol

胰岛移植(TX)的准备

  1. 倒置显微镜下,亲自挑选使用P200的pipetman和直吸管尖在一个100mm板的培养胰岛的胰岛细胞。
  2. 计数胰岛细胞的时间和传输到每个离心管(〜500胰岛细胞/管/鼠标)100。
  3. 允许小岛定居离心管底部。
  4. 绘制成P200 pipetman使用直薄壁吸管尖(130ul)胰岛沉淀。
  5. 放置在注射器尖端的硅胶管适配器。硅适配器插入PE50管材的长度。
  6. 杭pipetman的油烟机和磁带比在尖端的胰岛更高PE50管罩壁的一侧。这将允许只在吸管尖的小岛定居。
  7. 慢慢拨号pipetman迁入PE50管材的胰岛,小心不要驱逐从最终的胰岛细胞,转移到PE50管材的小岛。
  8. 请在PE50管斜面的扭结。在保持扭结,从pipetman断开的硅胶适配器管和安全与硅适配器扭结。
  9. PE50管材,扭结硅适配器管面朝下,将小岛,到15毫升锥形削减5毫升吸管和磁带超过5毫升吸管一边PE50管,以防止卷曲而离心PE50管材。
  10. 1000转离心PE50/15mL conicals和关闭离心机。 (切勿超过10个胰岛筹备工作准备一次)。
  11. 将管放置在冰上。

供移植用小鼠制备

  1. 麻醉小鼠isofluorane或氯胺酮/ Xylexene(见准备协议)。
  2. 麻醉后已采取的影响,刮胡子鼠标左键侧面。
  3. 擦拭皮肤,鼠标,中心,聚维酮碘拭子,然后与ETOH棉签擦拭过。
  4. 找到左肾(正好脾)。做一个小切口,在皮肤暴露腹膜。
  5. 做一个小切口暴露肾脏腹膜。保持切口小,将有助于保持肾脏的提出和暴露。
  6. 切口的两侧施加轻微的压力,提高肾脏或鼠标弹出。
  7. 保持肾湿润,申请用棉生理盐水棉签放倒。
  8. 使用注射器23或25号针头,右侧肾脏上的一个小的便笺,创建一个缺口在肾囊;不能过深或过大。

移植胰岛

注:当鼠标正在准备发射,第二人应该准备的汉密尔顿螺杆驱动胰岛移植注射器。

  1. 慢慢地将PE50管材的硅胶适配器管,同时保持在管扭结。
  2. 放置到硅适配器油管和地点到枪头“螺杆驱动”玻璃汉密尔顿注射器的硅胶适配器管PE50管的另一端。慢慢松开PE50管扭结,确保小岛不漏。
  3. 慢慢地促进胰岛提示PE50管使用的“螺丝钉”的机制,但保持在PE50管材的胰岛前一个小气泡。
  4. 进入尼克在肾脏,小心地将包膜下PE50管材,使一个小口袋。要非常小心,不挖出胶囊通过肾脏或穿刺。
  5. 它有助于保持与生理盐水庆大霉素浸泡棉花放倒棉签面积和胶囊湿润;干囊容易撕裂。
  6. 轻轻移动在各个方向上的油管,建立一个“口袋”休息的胰岛移植。记住重新申请额外的生理盐水,以保持该地区的湿润。
  7. 根据谁打开了鼠标划归补肾胶囊的PE50管材,第二的人,谁准备的鼠标和汉密尔顿移植注射器,人的方向会慢慢推进包膜下小岛,里面的“口袋”,背后PE50管材提供一个小的空气泡,直到所有的胰岛移植。
  8. 慢慢地将PE50管材,干用干的棉签区域,并仔细烧灼低热量的尼克。
  9. 放倒使用干燥的棉花拭子,确保所有的出血已经停止。一旦出血停止后,重新用无菌生理盐水湿润的肾脏,并轻轻地更换成腹膜前肾缝合和皮肤订书钉关闭鼠标。

关闭/鼠标复兴

  1. 关闭正在运行的W / C - 619毫米针用5-0丝线缝合针腹膜。
  2. 使用镊子一起绘制皮肤切口两侧。
  3. 副食皮肤连同2个或3个订书​​钉。
  4. 清洁皮肤的血液任何鼠标,使用棉花放倒拭子和盐度E.
  5. 鼠标立即与氟尼辛和丁丙诺啡皮下注射治疗。
  6. 鼠标放置在一个笼子里,放在加热垫或低于取暖灯,直到鼠标是完全积极的。
  7. 将在2个星期的皮肤订书钉。

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Discussion

该协议提供了一个干净,方便地提供糖尿病患者或正常小鼠肾被膜下胰岛细胞或细胞的实际和有效的选择。集中和造粒最终交付油管,用于移植的肾囊下细胞(PE50)的胰岛细胞或细胞的技术,提供了一个移植的细胞,而减少任何不必要的压力细胞或鼠标简单而有效的方法。

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Acknowledgements

加州大学旧金山分校糖尿病中心。美国国立卫生研究院加州大学旧金山分校DERC胰岛核心。 JDRF。 LIFESCAN公司强生公司。

Materials

Name Company Catalog Number Comments
Surgical gloves Fisher Scientific 11-394-95(sz)
Surgical scrub sponges Moore Medical 42940AK
Forceps Miltex Inc. 6-114 and 6-26 2 with teeth, 2 straight
Dissecting scissors n=2 Miltex Inc. 5-290
Oster razor (sz. 40 blade) Fisher Scientific 01-305-10B
4x4 Sterile gauze pads Moore Medical 08252AK
Povidone Iodine pads Moore Medical 08486AK
Alcohol pads Fisher Scientific 14-819-2
Heating pad Moore Medical 42508AK
23G or 25G needles 1" long
Cidex solution Moore Medical 07535AK for cleaning/santizing instruments
PE50 polyethylene tubing BD Biosciences 427411 PE50, 0.965mm O.D. x 0.58mm I.D.
Cidex + 28 day soln Moore Medical 35625AK
Silicone tubing Spectrum Chromat. 123732 5/32”OD x 1/32” ID
Instrument sterilizing container Moore Medical 39074AK
Straight Pipet Tips USA Scientific, Inc. 1111-0810 sterile
Glass syringe w/screw-drive Hamilton Co 1001
Cauterizing tool Roboz Surgical Instruments Co. RS230
Needle Holder Moore Medical 41-067
5-0 silk LOOK Surgical 754B for suture (6-C)
Cotton tipped swab Moore Medical
9mm autoclip stapler BD Biosciences 7630
9mm staples BD Biosciences 7631
9mm staple remover BD Biosciences 7637

DOWNLOAD MATERIALS LIST

References

  1. Tang, Q., Henriksen, K. J., Bi, M., Finger, E. B., Szot, G., Ye, J., Masteller, E. L., McDevitt, H., Bonyhadi, M., Bluestone, J. A. In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J Exp Med. 199, 1455-1465 (2004).
  2. Rulifson, I. C., Szot, G. L., Palmer, E., Bluestone, J. A. Inability to induce tolerance through direct antigen presentation. Am J Transplant. 2, 510-519 (2002).
  3. Lenschow, D. J., Zeng, Y., Hathcock, K. S., Zuckerman, L. A., Freeman, G., Thistlethwaite, J. R., Gray, G. S., Hodes, R. J., Bluestone, J. A. Inhibition of transplant rejection following treatment with anti-B7-2 and anti-B7-1 antibodies. Transplantation. Transplantation. 60, 1171-1178 (1995).
  4. Lenschow, D. J., Zeng, Y., Thistlethwaite, J. R., Montag, A., Brady, W., Gibson, M. G., Linsley, P. S., Bluestone, J. A. Long-term survival of xenogeneic pancreatic islet grafts induced by CTLA4lg. Science. 257, 789-792 (1992).

Comments

12 Comments

  1. Could you tell me which  type of Hamilton screw drive syringe be used?  Hamilton  Precision Syringe Pump PSD-² or PSD-3 or somethingelse?

    Reply
    Posted by: Anonymous
    September 25, 2008 - 5:21 AM
  2. We use the Hamilton #1001 syringe catalog# 81341.  Its a threaded plunger syringe that includes a screw drive plunger; $²59. Website: http://www.hamiltoncompany.com/syringes/syringesLS-Threaded.asp We then cut the back-end of a pipet tip to fit on the leur tip of the syringe.  This allows for the attachment of the silicone adaptor tubing to attach to the syringe and move your pellet of cells through the PE50 polyethylene tubing from Becton Dickenson #4²741.

    Reply
    Posted by: Anonymous
    September 25, 2008 - 12:18 PM
  3. Hi Dear, the kidney is located retroperitoneally, that means if you have chosen approach from backside, there is not any peritoneum to be cut and sutured.

    Reply
    Posted by: Anonymous
    October 20, 2008 - 3:46 AM
  4. I see smart people

    Reply
    Posted by: Anonymous
    October 20, 2008 - 1:13 PM
  5. Could you tell me what type of silicone adaptor tubing is used when it is attached to pipet tip? Is it commercially available?  

    Reply
    Posted by: Anonymous
    November 27, 2008 - 2:45 AM
  6. Hello, I can't seem to find the Syringe with the screw-drive on the Hamilton website, dŒs anybody have anymore information on this item? Thank you so much!

    Reply
    Posted by: Anonymous
    February 9, 2009 - 9:12 PM
  7. Hello! Thank you for this wonderfull and very instructive video! I was wondering if it is possible to save the video on a harddrive! If yes could you tell me how to do it Thank you a lot in advance   Evrim Jaccard PhD student  

    Reply
    Posted by: Anonymous
    February 26, 2009 - 12:42 PM
  8. Hello Evrim, Why don't you shoot me an email at nikitab@jove.com and we'll see what we can do. Cheers, Nikita

    Reply
    Posted by: Anonymous
    April 17, 2009 - 2:19 AM
  9. Hi Dr. Szot, Thank you for posting a very helpful video demonstrating islet transplant.  What do you think of isolating islet cells and culturing them overnight before transplantation the next day?  If you think this possible, do you recommend any particular culturing conditions (medium, temp, etc)? Thank you in advance, Omar

    Reply
    Posted by: Anonymous
    March 13, 2009 - 6:43 PM
  10. Thanks for sharing the impressive video.
    Would you like to tell me, in your experience, how long did the mice maintain their euglycemia status after receiving transplantation?

    Reply
    Posted by: Ruei-Ren W.
    July 16, 2009 - 4:52 AM
  11. Excellent work to teach the people how to do the surgery

    Reply
    Posted by: Anonymous
    November 5, 2009 - 1:23 PM
  12. Dear Librarian,
    I would like to request access to JoVE because I am a young scientist and study the problem of diabetes. Your help will be invaluable to me.
    Alibek Mereke
    Department of surgery, Kazakh National Medical university, Almaty

    Reply
    Posted by: Alibek M.
    January 9, 2012 - 9:50 AM

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