Name: a mouse model of vascularized heterotopic spleen transplantation for studying spleen cell biology and transplant immunity
Uploaded: 2019-06-11T15:00:00
Description: Watch this Scientific Journal Video about A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity at JoVE.com

作者感谢西北大学综合移植中心和费恩伯格医学院研究核心项目的资源和资金支持。具体而言, 流式细胞术和组织学服务分别由西北大学流式细胞术核心设施和小鼠组织学和表型实验室提供, 这两个机构都得到了授予罗伯特 h 的 NCI P30-CA060553 的支持。卢里综合癌症中心。我们感谢内特·埃斯帕扎先生校对了这份手稿。

本研究中的所有程序和动物使用都是根据西北大学内部动物护理和使用委员会 (IACUC) 批准的协议进行的。在这项研究中, 8 至10周龄的男性 CD45.2 和 CD45.1 小鼠 (均为 balb1 背景, 杰克逊实验室) 分别作为脾脏捐献者和接受者, 建立了同种异体脾脏移植模型。所有的动物都被安置在西北大学动物设施的无菌环境中。眼睛润滑剂应用于所有小鼠麻醉后, 以防止干燥。
1. 手术准备、麻醉和?...

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令人信服的证据表明, 脾衍生单核细胞在无菌炎症过程中发挥着重要作用, 如动脉粥样硬化19、急性缺血性脑20或肺损伤18, 以及心肌 isr 损伤和改造21,22,23。这些报告突出表明, 脾脏在许多慢性病中的作用被低估, 其中心血管疾病是重要的 (特别是考虑到它是全球头号杀手)。脾?...

脾脏是人体最大的继发性淋巴器官, 在免疫和造血系统中至关重要。它的功能主要是由两个形态上不同的隔间, 红色纸浆和白色纸浆1。红色牙髓是由网状纤维、网状细胞和相关巨噬细胞组成的静脉窦和脾脊髓的三维网状结构。这种独特的结构使红色的果肉作为一个有效的血液过滤器, 去除异物和旧的或损坏的红细胞。白色牙髓包括卵泡、边缘区和动脉周围淋巴鞘 (ALS), 是抗原捕获和处理、淋巴细胞归巢、转化、增殖和成熟2的重要部位。然而, 脾脏通常被认为是一个可有可无的器官, 因为其他淋巴器官, 如淋巴结, 也可以执行其一些功能, 脾脏的丧失通常不会导致死亡。因此, 脾切除术已被广泛用作脾损伤或良性血液疾病患者的一种治疗方法3。然而, 脾切除术的患者面临着一些长期的并发症。细菌感染是脾切除术4,5 的最佳公认并发症。最近, 压倒性的脾切除术后脓毒症已被认为是脾切除术的一个密集的并发症与高死亡率6。此外, 最近的流行病学研究表明, 脾切除术可能与心血管疾病的发生有关, 这表明脾脏的进一步生理功能仍有待探索 7,8。
脾脏自体移植和同种异体脾移植在临床上都得到了应用。目前, 脾脏自体移植通过植入部分的脾脏组织在大动量产生的袋被认为是唯一的可能性, 以保持脾功能后外伤性脾切除 9,10.然而, 这种手术的疗效是值得商榷的, 手术后并发症, 如脾组织无菌坏死和小肠梗阻, 由于术后粘连可能发生11。同种异体脾移植参与多脏器移植12。多脏器移植的临床证据表明, 同种异体脾移植可能在小肠同种异体排斥反应中起保护作用, 而不会引起移植物抗宿主病 (GVHD)12。然而, 关于同种异体脾移植作为多脏器移植成分的有益效果的文献仍然有限, 其潜在机制仍有待界定。2006年, Yair Reisner 等人报告说, 将没有 T 细胞的猪胚胎脾脏组织移植到小鼠身上可以治愈血友病 A, 这是一种没有引起 GVHD13的遗传性疾病, 支持脾脏移植具有治疗作用。某些疾病。因此, 有必要进一步研究脾脏移植的治疗潜力。
脾脏移植动物模型对于探讨脾脏免疫细胞在疾病进展中的不欣赏功能以及测试脾脏移植的潜在治疗效果具有重要价值。正如科恩14号所评论的那样, 自20世纪初以来, 已经记录了整个实验性的脾脏移植模型。1969年, coburn richard j. 和 lee 等人详细介绍了15、16 大鼠的脾脏移植技术。最近, Swirski FK 等人描述了一个小鼠脾脏移植17模型。与大鼠模型相比, 小鼠脾脏移植模型由于其固有的几个优点而更具吸引力。例如, 通过使用鼠标模型, 我们可以访问大鼠模型所无法获得的各种试剂。此外, 通过使用同源小鼠 (例如, 具有 cd45. NCD45.2 背景的小鼠), 同种异体脾脏移植可以跟踪脾细胞18 的命运、寿命和功能。在 Swirski FK 等人的工作基础上,我们进一步建立了这种简化和增强的小鼠脾移植方案。下面描述的协议以标准化的方式将可靠性和可行性结合起来, 可作为研究脾脏生物学和移植免疫的工具。

<table>
	<tbody>
		<tr>
			<td>Ketamine</td>
			<td>Wyeth</td>
			<td>206205-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Lloyd Laboratories</td>
			<td>139-236</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin solution</td>
			<td>Abraxis Pharmaceutical Products</td>
			<td>504031</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection grade normal saline</td>
			<td>Hospira Inc.</td>
			<td>NDC 0409-4888-20</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Pharmco Products Inc.</td>
			<td>111000140</td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoCare Small Animal ICU System</td>
			<td>Thermocare, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adson Forceps</td>
			<td>Roboz Surgical Instruments</td>
			<td>RS-5230</td>
			<td></td>
		</tr>
		<tr>
			<td>Derf Needle Holder</td>
			<td>Roboz Surgical Instruments</td>
			<td>RS-7822</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra Fine Micro Dissecting Scissors</td>
			<td>Roboz Surgical Instruments</td>
			<td>RS-5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-clip</td>
			<td>Roboz Surgical Instruments</td>
			<td>RS-5420</td>
			<td></td>
		</tr>
		<tr>
			<td>7-0 silk</td>
			<td>Braintree Scientific</td>
			<td>SUT-S 103</td>
			<td></td>
		</tr>
		<tr>
			<td>11-0 nylon on 4-mm (3/8) needle</td>
			<td>Sharpoint DR4</td>
			<td>AK-2119</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms CD45.2 antibody</td>
			<td>BD Bioscience</td>
			<td>553772</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms CD45.1 antibody</td>
			<td>BD Bioscience</td>
			<td>553776</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms CD11b antibody</td>
			<td>BD Bioscience</td>
			<td>557657</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms B220 antibody</td>
			<td>BD Bioscience</td>
			<td>553089</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms Ly6C antibody</td>
			<td>eBioscience</td>
			<td>48-5932-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms Ly6G antibody</td>
			<td>BD Bioscience</td>
			<td>561236</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms F4/80 antibody</td>
			<td>BD Bioscience</td>
			<td>565614</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms CD11c antibody</td>
			<td>BD Bioscience</td>
			<td>558079</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms CD3 antibody</td>
			<td>eBioscience</td>
			<td>48-0032-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms CD4 antibody</td>
			<td>BD Bioscience</td>
			<td>552051</td>
			<td></td>
		</tr>
		<tr>
			<td>Ms CD8 antibody</td>
			<td>BD Bioscience</td>
			<td>563786</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD&#8482; Fixable Violet Dead Cell Stain Kit</td>
			<td>Thermo Fisher</td>
			<td>L34955</td>
			<td></td>
		</tr>
	</tbody>
</table>

a mouse model of vascularized heterotopic spleen transplantation for studying spleen cell biology and transplant immunity

脾脏是一个独特的淋巴器官, 在免疫和造血系统的稳态中发挥着至关重要的作用。已接受脾切除术的患者, 无论沉淀原因如何, 都容易出现压倒性的脾切除术后感染, 并经历深静脉血栓形成和恶性肿瘤的风险增加。最近, 流行病学研究表明, 脾切除术可能与心血管疾病的发生有关, 这表明脾脏的生理功能尚未得到充分认识。本文介绍了一种小鼠血管化异位脾移植模型, 该模型不仅可用于研究脾脏免疫细胞亚群在不同生物过程中的功能和行为活动, 而且可以作为检测的有力工具脾脏移植治疗某些疾病的潜力。该模型的主要手术步骤包括供体脾收获、供者原生脾脏切除和脾移植血运重建。使用同源小鼠菌株 (例如, 具有 cd45. 半 CD45.2 背景的小鼠), 我们观察到, 在同种异体移植后, 捐献者衍生的脾淋巴细胞和骨髓细胞早在术后第1天就从移植物中移出, 并与多种类型的接收细胞的涌入, 从而产生一个独特的嵌合体。 尽管技术相对具有挑战性, 但这一过程可以用 & gt;90 的成功率来完成。该模型允许跟踪脾细胞在稳定状态下和脾脏移植后的疾病设置中的命运、寿命和功能, 从而提供了一个很好的机会来发现脾脏衍生免疫细胞的独特作用。不同的疾病过程。

有经验的显微外科医生可以在90分钟内完成小鼠脾脏移植的整个过程。我们的实验室已经在小鼠身上进行了100多次脾脏移植。成功率超过 90%, 定义为受者小鼠和脾脏移植到术后日 (POD) 1 或 POD 7 (我们的研究终点) 的生存率。脾脏移植的存活得到了脾脏细胞宏观外观和流式细胞仪分析的证实。根据我们的经验, 流式细胞仪分析 (liveydead 细胞活力检测) 是非常敏感的, 以确定脾脏?...

Watch this Scientific Journal Video about A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity at JoVE.com

A Mouse Model of Vascularized Heterotopic Spleen Transplantation for Studying Spleen Cell Biology and Transplant Immunity

血管化异位脾移植小鼠模型在脾细胞生物学和移植免疫研究中的应用

该协议详细介绍了血管化异位脾移植小鼠模型的手术步骤, 这是一种技术上具有挑战性的模型, 可作为研究脾脏细胞命运和寿命的有力工具, 而脾脏是不同脾脏的机制细胞群在疾病进展, 和移植免疫。

The spleen is a unique lymphoid organ that plays a critical role in the homeostasis of the immune and hematopoietic systems. Patients that have undergone ...

脾脏移植的小鼠模型为揭示脾脏细胞在炎症性疾病中局部和全身免疫反应的作用提供了绝佳的机会。该协议的主要优点是，通过使用致癌小鼠，脾脏移植可以检查脾脏细胞的命运、寿命和功能。在开始手术之前，在手术平台上放置一个无菌的一次性窗帘，并确认麻醉供体小鼠对手趾捏缺乏反应。剃毛从整个腹部区域，并放置鼠标在手术显微镜下在6至10倍的放大率无菌手术平台。用酒精准备垫对裸露的皮肤进行消毒，用手术胶带固定四肢。通过三到四厘米的中线垂直皮肤切口进入腹部，从到 xyphoid 过程。使皮肤血管发白，将切口延伸到两侧的侧腹部，以便更好地暴露。使用无菌棉签，将肠子移动到腹部右侧以暴露脾脏，并使用无菌低温烧灼烧灼附于脾脏的短胃静脉。将一块无菌纱布浸泡在脾脏上，用37摄氏度的盐水浸泡，以保持其湿润，分离和调动入口静脉从胰腺组织。如所证明，将门户静脉的分支进行拉起。将缝合线从脾脏静脉的入口静脉周围放置，然后将脾脏翻转到右侧，以暴露腹腔躯干的主动脉和脾动脉。解剖和调动主动脉腹腔动脉，并在主动脉周围缝合到腹腔动脉附近。将 100 个国际肝素单位注入劣质的文纳卡瓦或 IVC 中， 使全身肝素化。三分钟后，将主动脉的肝动脉和胃动脉与腹腔动脉连接。从腹主动脉到腹腔躯干，用10毫升的4摄氏度肝素盐水将全身透水。收集脾脏移植和块与相关的动脉腹腔脾脏片段在门户静脉以及脾脏静脉和胰腺组织的一小部分。然后在五毫升四摄氏度的盐水中保存移植物。手术区域准备后，通过中线切口进入接收者腹部，就像为捐赠者一样，通过连接脾脏和动脉来去除接受者的原生脾脏。用37摄氏度浸泡的纱布盖住肠组织，小心地将肠子移动到左侧。用两个四毫升微血管夹解剖和合接 IVC 中下动脉大动脉的腰椎，并交叉夹紧 IVC 中的下动脉。将11-0尼龙缝合线穿过骨道，并缩回容器，以便创建带微切口的椭圆肌。使用 30 量针刺穿 IVC 以创建椭圆形静脉切除术，并延长开口到供体入口静脉的长度。用500微升的肝素盐水冲洗大动脉和 IVC，清除血管中的血内血或血块，将供体脾脏移植物放入接受者腹部的右侧。仔细识别供体大动脉袖口和入口静脉。在确认容器没有扭曲后，用四摄氏度的盐水浸泡纱布盖住脾脏移植物。使用两个 11-0 留缝合线，将供体大动脉袖口连接到接受者动脉切除术的近位和近侧顶点，并在供体大动脉袖口和接受者耳管切除术前壁之间用两到三口连续 11-0 尼龙缝合进行心肺病。将脾脏移植物转向接受者左侧，在供体大动脉袖口和接受者耳皮后壁之间进行心肺病。在供体入口静脉和接受者 IVC 之间进行静脉动脉瘤，每侧有四到五口连续缝合。先缝合容器流明内的后壁，然后使用相同的缝合线关闭前壁。在 IVC 外的下角放置一个结，完成结切除术。现在释放容器夹，并使用无菌棉签棉布棉布，直到脾脏颜色恢复。以连续图案的 5-0 合成可吸收的 Vicryl 缝合线关闭腹部，并在中断的图案中用 5-0 尼龙缝合线关闭皮肤层。然后将250微升热盐水下皮注射到四个不同的位置，并在30摄氏度的温度控制培养箱中加热小鼠，在手术后最初几个小时进行监测，直到动物恢复足够的意识，转移到家庭笼子。移植后脾脏异体的第一天和七天，脾脏异丙酮的血氧素和eosin显示，脾脏异体结构在术后的第一周保持不变。流动细胞测量分析，研究移植后供体脾细胞迁移，结果表明，约50%的脾细胞是移植后一天产生的供体，约46%是接受者。移植后七天，供体衍生的白细胞约占脾细胞总数的32%，而接受者衍生的细胞增加至约53%，供体白细胞早在第一天就迁移到淋巴结、血液和骨髓中，并至少维持到第七天，产生一种独特的奇美拉，对脾细胞贩运研究有价值。使用转基因敲击小鼠作为脾脏捐赠者将允许调查脾脏细胞衍生中介或关键信号通路在疾病过程中的作用。该模型可能是探索脾脏细胞群的反应病原体，损伤，炎症或移植排斥机制的有力工具。

Research

JoVE Journal

Medicine

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

主动脉原位移植大鼠模型，研究慢性血管病变的发展

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

科研

医学

A Mouse Model of in Utero Transplantation

A Mouse Model of in Utero Transplantation

一个小鼠模型在子宫内移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

平移血管复合同种异体移植（VCA）研究一种改进的异位猪后肢移植模型

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma ...

A Modified Method for Heterotopic Mouse Heart Transplantion

对于异位小鼠心脏移植技术的一种改进方法

Mice are often used as heart transplant donors and recipients in studies of transplant immunology due to the wide range of transgenic mice and reagents ...

Murine Heterotopic Heart Transplant Technique

小鼠异位心脏移植技术

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

肺动脉瓣使用异位心脏移植小鼠模型移植

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

一种新的显微模型异位，整块胸壁，胸腺，和心脏移植小鼠

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

Orthotopic Hind Limb Transplantation in the Mouse

原位后肢移植中的鼠标

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

异位自体肾移植的猪模型：有步骤，分步协议

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...