小鼠皮肤移植

Biology

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Summary

异体皮移植是一个标准的模型来检测主机的T细胞应答的MHC不同的捐助抗原。此视频文章执行BALB/c--> C57BL / 6皮肤移植的每个步骤提供了一个可视化教程。

Cite this Article

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Garrod, K. R., D. Cahalan, M. Murine Skin Transplantation. J. Vis. Exp. (11), e634, doi:10.3791/634 (2008).

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Abstract

皮肤移植的最严格和最不发达国家在技术上具有挑战性的车型之一,是一个标准的方法来检测主机的T细胞应答的MHC不同的捐助抗原。此视频文章的目的是向观众提供了一步一步的视觉使用皮肤移植小鼠模型示范。该协议分为5个主要部分组成:1)皮肤收获捐助; 2)准备移植; 3)皮肤移植; 4)绷带拆除和移植排斥反应的监测; 5)有用的提示。一旦精通,程序本身应采取<10分钟来执行。

Protocol

实验开始之前:

  • 获取IACUC批准
  • 消毒手术器械和Q -秘诀

一,采收供体组织

  1. 安乐死捐助鼠标(按照机构的SOP)
  2. 湿漉漉的头发从耳根用酒精回
  3. 收获基地耳捐助皮肤
  4. 把组织上的冰板
  5. 轻轻拉开分割的耳朵:捏腹侧和背侧组织双方在基地与鼠齿钳耳;组织;浮动背侧组织,内部表面向下,在PBS冰;丢弃胶原腹皮瓣

二。为移植做准备

  1. 麻醉鼠标使用批准的程序
  2. 管理镇痛术后疼痛缓解(即0.05-0.1毫克/克Bupernex IP)
  3. 将鼠标在卧位
  4. 执行脚趾捏,以确保动物有足够的镇静剂
  5. 用酒精和湿漉漉的头发剃胸部
  6. 用酒精棉球擦拭面积清洁

三。皮肤移植

  1. 请〜1厘米的切口:用鼠齿钳帐篷的皮肤和削减〜0.25-0.5厘米以下钳
  2. 删除捐助皮肤从PBS,植骨床上面的地方(面临植骨床的耳朵皮肤表面内部),并删除多余的PBS用无菌棉条
  3. 仔细修剪多余的捐助使皮肤移植在于断然植骨床内(确保移植边缘卷曲下,移植到收件人的躯干部皮肤不解酒)
  4. 在无菌绷带包裹收件人(与非粘性纱布段放置在皮肤移植)
  5. 确保绷带是不是太紧(评估动物的呼吸),武器可以自由移动
  6. 用一个2-0丝线缝合的安全绷带
  7. (下加热,直到动物灯放置在清洁的笼子里的动物有足够的恢复从麻醉中)
  8. 监视器收件人1-2/day遇险的迹象和管理镇痛止痛的需要

四。绷带拆除(移植后6-7天)

  1. 麻醉鼠标使用批准的程序
  2. 剪切和删除丝线缝合
  3. 使用钝年底剪刀,绷带(确保不破坏移植)通过精心切割,轻轻取出
  4. 返回鼠标清洁笼
  5. 移植〜8小时后检查包扎去除scabbing或收缩的迹象。如果存在,可能已经失败移植血管,应被认为是技术故障。
  6. 每日监测排斥反应的迹象(通常定义为〜80%的捐助组织坏死)
  7. 异体皮移植之间的D8 - 12 - OP后通常会拒绝

五,帮助提示

  1. 收获捐助组织接近耳朵的基础:1)最大限度地提高移植物的大小,和2)提供一个不平衡的腹侧,将有利于分裂的悬
  2. 请务必使用一个新的刀片剃须收件人
  3. 如果是给定的IP镇痛,管理移植前的程序,以尽量减少术后处理(最大限度地减少扰乱了移植的机会)
  4. 如果可能的话,除去手套,然后用绷带。手套往往坚持绷带和复杂的应用程序
  5. 放置在一个单一的缝合包扎结束,将最大限度地减少过早拆除
  6. 裹绷带严格,不允许和评估前放置缝合呼吸。如果包扎过紧,动物就会停止呼吸。如果太松绷带,动物将其删除。

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Discussion

在这个视频文章表明,皮肤移植模型是一个监测异体T细胞反应的快速和简便的方法。异体皮移植,具有广泛的应用,并已被用来检测各种免疫抑制剂和耐受的协议,以及调查各种阻断抗体的生物效应的效率。

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Acknowledgements

美国国立卫生研究院免疫学练金的T32人工智能60573(KRG)和美国国立卫生研究院授予通用汽车41514(MDC)

Materials

Name Company Catalog Number Comments
Micro-dissecting forceps (curved) Sigma-Aldrich F4142
Rat-tooth forceps (1x2 teeth, straight) Fine Science Tools 11053-10 n=2
Strabismus scissors (blunt end) Fine Science Tools 14074-11
Iris forceps (curved) Fine Science Tools 11065-07
Spring scissors (angled) Fine Science Tools 15006-09
Halsey micro needle holder Fine Science Tools 12500-12
10 cm plates Corning 430167
PBS Cellgro 21-040-CV
Bupernex (Buprenorphine hydrochloride) Reckitt Benckiser 517201
Insulin syringes (29 Ga) Monoject 8881 600145
Q-tips Solon Manufacturing Company 362
Single edge razor blades (#9) Smith & Nephew Inc. 67-0238
Silk suture (2-0; PS-2) Ethicon Inc. 583H

DOWNLOAD MATERIALS LIST

Comments

20 Comments

  1. Kym, I would really be interested to see a histologic profile of the post-op period - do you have the respective images?
    Thank you in advance,
    Peter

    Reply
    Posted by: Anonymous
    January 22, 2008 - 4:34 PM
  2. Dear Peter- Thanks for the great suggestion. I've contacted JoVE to see if a section can be added to the article containing histo pics from allo- and syn- transplants.

    Reply
    Posted by: Anonymous
    January 22, 2008 - 7:33 PM
  3. The connecting speed is too slow, it spends loog time to watch the whole video. Can we download the video?

    Reply
    Posted by: Anonymous
    March 28, 2008 - 9:35 AM
  4. Hi Merlin. I am sorry you are having trouble.  Is it your connection that is slow or ours?  Please use www.speedtest.net to test your connection.  If the problem is on your end, we are now working on a solution to help people with slow connections.  If you would like to discuss this, please send me an email directly to support@jove.com and I'll see what may be the problem.

    Reply
    Posted by: Anonymous
    March 28, 2008 - 11:09 AM
  5. Hi,nikitab. Thank your help. I am from China, I test my connection, the connection is slow from USA to my city. could you tell me how to fix this problem?

    Reply
    Posted by: Anonymous
    March 28, 2008 - 10:04 PM
  6. If you click the pause pause/play button, the video will buffer and you should be able to play it without interruptions after some time.  Hope tihs helps!

    Reply
    Posted by: Anonymous
    March 28, 2008 - 10:21 PM
  7. Merin, just wanted to let you know that we've upgraded the video player.  How are the videos playing now?

    Reply
    Posted by: Anonymous
    May 15, 2008 - 6:34 PM
  8.  wow ive learned alot from this can you tell me more about this? is it different from human skin transplants? there is alot going on with stem cell, is this simular, this is intresting, would this maby help people that have bad skin burns?

    Reply
    Posted by: Anonymous
    February 3, 2009 - 11:16 PM
  9. hello this is interesting, is this simular to human skin transplants? dŒs this help people with bad skin burns?  theres alot going on with stem cell research is this simular? i have learned alot from this vidio, i wait for your answers.

    Reply
    Posted by: Anonymous
    February 3, 2009 - 11:32 PM
  10. Hi, Kym, I learn the technique of ear-skin graftting through the video. Well, in the articles about skin graft in mice, most of them just use the method of back-back or tail-back skin transplantation. And do you provide some articles about ear-back skin graft to me? Thank you very much.
    cai

    Reply
    Posted by: Anonymous
    July 9, 2009 - 7:37 PM
  11. beautiful video. we use a similar technique at the Diamantina Institute, but I got some handy hints watching your technique. thanks.

    Reply
    Posted by: Anonymous
    August 5, 2009 - 1:41 AM
  12. Are donor and recipient mice females or males?

    Reply
    Posted by: Anonymous
    September 24, 2009 - 12:54 PM
  13. Hi Kym. I was wondering if you or your colleges transplant embryonic skin that way, or if there has to be special protocol for that?

    Reply
    Posted by: Anonymous
    January 21, 2010 - 4:29 PM
  14. Hi Kim,
    we do skin grafts and we have problems with the bandage. I use band-aids (the ones sold in the regular pharmacy for cuts and bruises) and mice chew them off as soon as they weak up from anesthesia. I do not see in your materials list what kind of bandage you use. Would you please tell me where do you buy it and the catalog number? I would really appreciate.
    best regards
    sg

    Reply
    Posted by: Anonymous
    May 28, 2010 - 3:03 PM
  15. why i can't see it? is it because the subscription lock? Our school don't have this database. How can i see it ? is it mean that that i need to buy it ?

    Reply
    Posted by: jin l.
    October 16, 2011 - 8:02 AM
  16. Access to this article is only available through an institutional subscription. Please contact your Librarian and include me on cc, and we will arrange a ² weeks FREE trial to the entire institution. Not only will this give you access to this article but will provide you with the opportunity to access other content.

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    Reply
    Posted by: Anonymous
    October 16, 2011 - 6:40 PM
  17. I am very sorry that i reply so late. At first, i try to contact you through email, but it was rejected by the recipient domain.
    I am from China and our school librarian didn't have this service now, so i only to contact you, and thank you to arrange a free trial for me. Please tell me what should i do to apply the free trial, you can send the details to me. If i can't receive your email. i will post message on jove.thank you again.
    Best wishes
    jin liu
    Chengdu, China. Chengdu medical college
    Sichuan Province, Chengdu Chengdu City Jinniu District Road No. 601

    Reply
    Posted by: jin l.
    October 19, 2011 - 9:37 PM
  18. Dear Jin,
    I have forwarded your response to our account manager in China. She will be in touch in due course. Thank you for your interest in JoVE.

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    Reply
    Posted by: Anonymous
    October 20, 2011 - 12:14 PM
  19. thank you!

    Reply
    Posted by: jin l.
    October 21, 2011 - 9:37 AM
  20. Hi,Kym, I am doing mouse ear skin grafts recently, I want to ask did mice ear cartilage layer, in the video is the inner or outer ear skin transplantation.Thank you very much!

    Reply
    Posted by: Anonymous
    May 14, 2017 - 4:17 AM

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