This work was supported by SNF grants PPOOA-_119204 and PPOOP3_144918 to S.W.R. We thank E. Palmer and B.T.H. Hausmann for mice and technical expertise.

在这个视频发布和协议，所有动物的程序按照批准由州局巴塞尔城市州，瑞士动物的协议进行的。执行在无菌条件下尽可能的所有程序。 外科1。准备<ol><li>高压灭菌所有的手术器械，使用前纱布。 </li><li>热保暖垫，并组织手术器械上的表 （表材料/方法 ）。 </li><li>打开手指带绷带。应用凡士林的伤口垫用棉签（确保纱布完全覆盖）。 </li><li>称取小鼠和管理镇痛丁丙诺啡0.05毫克/公斤体重。注：这将立即移植后提供镇痛。 </li></ol> 2，准备的尾部皮肤移植<ol><li>每二氧化碳窒息和D安乐死供体小鼠（ 例如 IA BM12）isinfect尾部与前切除术消毒液。 </li><li>定位尾部的黑色条纹倒在​​一个干净的塑料板，切纵向贯穿中间路线，做一个肤浅的切口仅累及皮肤层。 </li><li>拉过使用镊子夹住尾巴的皮肤。 </li><li>把尾部皮肤在10cm的细胞培养皿填充用10ml HBSS中。 </li></ol> 3，外科手术<ol><li>通过吸入以透明感应箱3％的异氟烷溶液麻醉受体小鼠（ 例如，C57BL / 6）。注意：这需要3-5分钟的鼠标，达到手术麻醉平面。使用上的眼睛兽医药膏，以防止肌肤干燥，同时在麻醉下。 </li><li>将鼠标放在暖垫，并通过一个口罩和驴由脚趾捏适用1.5％异氟烷每个动物的条件反射。 </li><li>剃接受者小鼠的背部的网站（ 例如，C57BL / 6），用干纱布SW去除污染的毛AB。 </li><li>消毒剃部位无菌解决方案移植。 </li><li>捏在背部中间/右现场镊子皮肤和使用弯曲剪刀，使约1×0.6厘米的圆形切口。 </li><li>如果发生出血，然后清洗伤口，用无菌棉签。 </li><li>评估该切口的大小和切断从使用手术刀尾部皮肤的估计大小的移植。 </li><li>修剪边角圆的移植。 </li><li>加载移植上​​的手术刀，干多余的HBSS在无菌棉签，并将其放置在受体小鼠的移植床。 </li><li>避免供体和宿主皮肤的重叠和应用上的接触区域组织粘合剂完全。 </li><li>应用无石膏鼠标左右腰部皱褶。 </li><li>适用于粘接非弹性绷带来固定石膏鼠标（2毫米移位）。 </li><li>切开以约3毫米的绷带的上腹部站点的剪刀。注意：这是为了避免TR鼠标的牙齿在恢复阶段apping。 </li><li>取出麻醉口面罩，并保持移植的小鼠在加热垫，直到清醒。 </li><li>返回小鼠移植到自己的笼子里，只有当完全康复，并评估其流动性，以确保绷带是不是太紧张。 </li><li>继续进行下一个手术前消毒用70％乙醇所有的外科手术器械。 </li></ol> 4，术后护理<ol><li>添加含扑热息痛的糖浆的饮用水（4毫克/毫升），并管理它7天。 </li><li>返回鼠标的动物房一旦恢复。 </li><li>密切留意老鼠的临床症状包括抑郁症或其他行为改变（饮食或不动），外观异常（缺乏疏导）或姿势（PILO勃起或弯腰驼背的姿势），当时的绷带到位（1周）。 </li></ol> 5，去除绷带（6-7天移植后） <ol><li> ANESthetize小鼠通过吸入以透明感应框（见上文）3％的异氟烷​​溶液。 </li><li>切绷带和石膏与动脉剪刀，小心地将其删除。注意：避免拉伸移植部位。 </li><li>监测移植小鼠的临床症状（见4.3），得分为抑制皮肤移植（ 表1）。注：该接枝评分系统采用的尾部皮肤较背侧皮肤不同发质的优势。移植和排斥的外观分为3级。 C57BL / 6小鼠免疫功能的异体皮（MHC II类错配， 例如，爱荷华BM12）移植物通常在12-15天移植后（自己的数据和15-20）拒绝。 </li></ol>

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The authors declare that they have no competing financial interests.

皮肤移植是一种广泛使用的方法为研究抑制和容忍依赖于T细胞。自成立以来的皮肤移植模型中，几个修改和变化都得到了应用。在描述的过程中，使用麻醉气体（异氟烷）进行IA BM12尾部皮肤移植。使用气体麻醉的降低执行和小鼠恢复，从而减少了应力对移植小鼠的时间。该程序使用组织粘合剂来固定尾部皮肤，这容易卷曲超过耳或背皮肤组织。此外，它弄平切割和修整的边缘，因为这降...

固体器官，如皮肤，心脏和肾脏移植是目前在全世界的医疗实践1的标准程序。成功移植的器官可以通过激活受体的免疫系统，其中确认捐助的主要组织相容性抗原被拒绝。因此移植的患者需要使用免疫抑制药物治疗2。异体皮移植在小鼠体内建立了梅达沃和他的同事于1955年，是识别后世誉为主要组织相容性复合体（MHC）I类和II目标分子的帮助。从那以后，皮肤移植模型得到了不断的修改和调整，研究T细胞亚群的作用和抑制移植排斥2-4化学和生物干预的相关性。从耳朵和躯干的皮肤都比较困难的准备，而且更容易受到缺氧和坏死较尾皮5;然而，移植过程是类似的。在另外的尾部皮肤移植物的监测是很容易由于皮肤的特征头发纹理。 本文提供的详细步骤MHC II类错配尾部皮肤移植，允许的CD4 + T细胞介导的移植排斥反应和耐受小鼠不同方面的研究。在MHC II类分子IA b将天然三点突变（称为IA BM12）6-9是足以引起排斥反应的皮肤移植物在C57BL / 6小鼠8。在IA BM12分子激活CD4 + T细胞与各种αβT细胞受体（TCR）从C57BL / 6小鼠链，其中Vα2Vβ8-TCR特异性T细胞，以产生一个TCR转基因小鼠10识别。 Vα2Vβ8-TCR特异性T细胞的过继转移已被用于建立一个排斥模型在免疫缺陷C57BL / 6的Rag2 - / -移植与IA BM12皮肤老鼠。 捐赠者和接受者之间的基因差异影响移植接受和拒绝的结果。有不同类型的移植：自体移植是从接收者个人自身移植; syngrafts和移植物分别从基因上相同和基因无关个体移植。不同的异体器官移植的接受已经证明了患者和小鼠模型11,3,4化学和生物干预。在一个基本的方法，抗-CD3抗体处理的C57BL / 6小鼠表现出IA BM12尾部皮肤（未发表数据），生存期延长。 CD4 +和CD8 + T细胞移植受体小鼠前的枯竭导致接受MHC I类和II不匹配的移植（修订版12）。有趣的是，抑制皮肤移植取决于CD4 +的存在</sup&gt; T细胞（版本12）。在此模型中，通过阻断共刺激分子的抗体或抑制与调节性T细胞靶向不同的免疫细胞之间的特异性相互作用可能诱导耐受（未发表数据）。事实上，无论阻断CD40和CD28分子导致了长期的皮肤移植耐受13,14。 相比于其它器官的移植尾部皮肤移植容易进行并易于监测。此外，尾部皮肤移植很容易准备，而且不太容易受到缺血较其他皮肤组织。而相比之下，注射麻醉剂，移植过程中使用麻醉气体（异氟烷）的缩短两者的程序和收件人的恢复时间。尾部皮肤移植，这可能会导致不完全的伤口愈合，移植物排斥的卷曲，可以防止通过应用组织粘合剂。此外，IA BM12尾部皮肤移植模型的专门激活的CD4 <sup&gt; + T细胞（相同遗传背景）两个免疫和免疫缺陷小鼠促进结果的解释。 本协议描述了一个可靠，重现性好，易于监控的小鼠模型，允许化学和生物干预。该模型的目的是调查排斥和耐受诱导的尾部皮肤移植手术。

<table border="0" cellpadding="0" cellspacing="0" width="831">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Name of Material/ Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:231px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine Standard solution</td>
			<td>Mundipharma</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton swab</td>
			<td style="width:183px;">Carl Roth GmbH</td>
			<td style="width:119px;">31025060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Dafalgan , UPSA</td>
			<td>Bristol Myers Squibb SA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hansaplast Finger Strips</td>
			<td>Beierdorf AG</td>
			<td style="width:119px;">REF.76861</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histoacryl Tissue adhesive</td>
			<td style="width:183px;">Braun</td>
			<td style="width:119px;">REF.1050052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leukotape classic , 2cm x 10 m</td>
			<td>BSN Medical SAS</td>
			<td>REF.02204-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS, Phosphate-Buffered Saline, pH 7.4</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:119px;">10010015</td>
			<td>GIBCO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile gauze, 5 x 5cm, 8ply</td>
			<td>MaiMed GmbH</td>
			<td>21010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical instruments&#160;</td>
			<td style="width:183px;">FST</td>
			<td style="width:119px;">11003-12</td>
			<td>Narrow pattern forceps,&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:183px;"></td>
			<td style="width:119px;">14095-11</td>
			<td>Fine iris scissor curved,</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:183px;"></td>
			<td style="width:119px;">14094-11</td>
			<td>Fine iris scissor&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:183px;"></td>
			<td style="width:119px;">14010-15</td>
			<td>Mayo scissors,</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:183px;"></td>
			<td style="width:119px;">14080-11</td>
			<td>Artery scissors ball tip 11.5 cm&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:183px;"></td>
			<td style="width:119px;">11021-14</td>
			<td>Tissue forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Surgical Blade No.20</td>
			<td style="width:183px;">Swann-Morton LTD</td>
			<td style="width:119px;">3006</td>
			<td>Carbon Steel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Surgical blade Handles</td>
			<td style="width:183px;">Swann-Morton LTD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe, 1ml</td>
			<td style="width:183px;">ARTSANA</td>
			<td style="width:119px;"></td>
			<td>disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Temgesic , Buprenorphine</td>
			<td style="width:183px;">ESSEX Chemie AG</td>
			<td style="width:119px;"></td>
			<td>0.3mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Tissue Culture dishes 10 cm, 60.1 cm2</td>
			<td style="width:183px;">TPP</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Vaseline</td>
			<td style="width:183px;">Vifor SA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Warm pad</td>
			<td style="width:183px;">Solis</td>
			<td style="width:119px;"></td>
			<td>Type 223</td>
		</tr>
	</tbody>
</table>

transplantation of tail skin to study allogeneic cd4 t cell responses in mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

在第一种方法中的C57BL / 6小鼠移植IA BM12同种异体移植物和IA b syngrafts。拆除绷带后，移植物伤口愈合的迹象明显没有在C57BL / 6小鼠（ 图1A）的接触带的闭合。拆除绷带后，CD4 + T细胞介导的炎症导致的坏死区域的外观（红点）和内移植（ 图1B-C，实心）后13天C57BL / 6小鼠抑制IA BM12移植，而syngrafts被容忍长达100天（ 图1B-C，空心符...

Watch this Scientific Journal Video about Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice at JoVE.com

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

transplantation-tail-skin-to-study-allogeneic-cd4-t-cell-responses

Research

JoVE Journal

Immunology and Infection

12.2K 次观看.  University of Basel and University Hospital Basel. Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.