Name: induction of murine intestinal inflammation by adoptive transfer of effector cd4+cd45rbhigh t cells into immunodeficient mice
Uploaded: 2015-04-21T13:00:00
Description: Watch this Scientific Journal Video about Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice at JoVE.com

这项工作是由美国胃肠病协会（AGA）研究学者奖和克罗恩和美国（CCFA）职业发展奖（以ENS），美国国立卫生研究院NIDDK F30 DK089692（以ECS）结肠炎基金会，以及北卡罗莱纳州中心大学的胃肠生物学支持与疾病格兰特P30 DK34987（组织学核心）。在UNC流式细胞仪核心设施是由NCI中心的核心支持格兰特（P30CA016086）的UNC莱恩伯格综合癌症中心支持的一部分。我们感谢卢克B.博斯特从兽医北卡罗莱纳州立大学对他的帮助与组织病理学分析和免疫组化。

注：确保所有的动物协议通过并符合机构动物护理和使用委员会（IACUC）的规定和国家研究委员会的指南实验动物的护理和使用的批准。供体小鼠可以是男性或女性，但受体小鼠应该是雄性。如果女性收件人使用，供体小鼠必须是女性5。使用常规的，非无菌床上用品和非酸化水中维持菌落，因为这些可能会影响肠道菌群，并且因此，小鼠5,6的结肠炎的表型。 <p class="jove_t...

<ol>
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在这里，我们描述了一个一步一步的协议，通过过继转移CD4 + CD45RB + T细胞的免疫缺陷小鼠诱导小鼠结肠的炎症。我们使用的C57BL / 6供体脾和同系RAG1 - / -受体小鼠，尽管其它菌株（ 例如 ，BALB / c小鼠，129S6 / SvEv，非肥胖糖尿病（NOD））免疫缺陷的和遗传模型（ 例如 ，SCID， 的Rag2 - / - ），也可以使用4,14-16。它是公认的背景品系?...

The inflammatory bowel diseases (IBD) Crohn&#8217;s disease and ulcerative colitis result from an incompletely defined and complex interaction between host immune responses, genetic susceptibility, environmental factors, and the enteric luminal contents1. Recent genome-wide association studies report associations between immune cell regulatory genes and IBD susceptibility2,3. Both innate and adaptive immune cell intrinsic genes are represented in these studies, indicating a central role for these cell populations in IBD pathogenesis.
There currently exist more than 50 animal models of human IBD. While no one model perfectly phenocopies human IBD, many are useful for studying various aspects of human disease, including disease onset and progression and the wound-healing response. In the method described here, intestinal inflammation is initiated with syngeneic splenic CD4+CD45RBhigh T cell adoptive transfer into T and B cell deficient recipient mice4. The CD4+CD45RBhigh T cell population contains mainly na&#239;ve T cells primed for activation that are capable of inducing chronic small bowel and colonic inflammation. This method allows the researcher to modify key experimental variables, including both innate and adaptive immune cell populations, to answer biologically relevant questions relating to disease pathogenesis. Additionally, this method provides precise initiation of disease onset and a well-characterized experimental time course. This permits the kinetic study of clinical features of disease progression in mice. Intestinal inflammation induced by this method shares many features with human IBD, including chronic large and small bowel transmural inflammation, pathogenesis driven by cytokines such as TNF and IL-12, and systemic symptoms such as wasting5. Thus, it is an ideal model system for studying the pathogenesis of human IBD.
The method here describes in detail the protocol for inducing experimental colitis by adoptive transfer of CD4+CD45RBhigh T cells into Rag1-/- mice. We discuss key technical steps, expected results, optimization, and trouble-shooting. We address the required elements for the successful development of this murine model of intestinal inflammation for research purposes.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Name of Reagent/ Equipment</td>
			<td >Company</td>
			<td >Catalog Number</td>
			<td >Comments/Description</td>
		</tr>
		<tr >
			<td >10x PBS</td>
			<td >Gibco</td>
			<td >14200075</td>
			<td></td>
		</tr>
		<tr >
			<td >12x75mm round-bottom tube</td>
			<td >Falcon</td>
			<td >352052</td>
			<td></td>
		</tr>
		<tr >
			<td >15 ml conical</td>
			<td >Corning</td>
			<td >430790</td>
			<td></td>
		</tr>
		<tr >
			<td >26g x 3/8 Needle</td>
			<td >BD Biosciences</td>
			<td >305110</td>
			<td></td>
		</tr>
		<tr >
			<td >50 ml conical</td>
			<td >Corning</td>
			<td >430828</td>
			<td></td>
		</tr>
		<tr >
			<td >70 um Cell Strainer</td>
			<td >Fisherbrand</td>
			<td >22363548</td>
			<td></td>
		</tr>
		<tr >
			<td >BD IMagnet</td>
			<td >BD Biosciences</td>
			<td >552311</td>
			<td></td>
		</tr>
		<tr >
			<td >&#946;-mercaptoethanol</td>
			<td >Thermo Scientific</td>
			<td >35602</td>
			<td></td>
		</tr>
		<tr >
			<td >CD4-FITC IgG2b</td>
			<td >eBioscience</td>
			<td >11-0041</td>
			<td></td>
		</tr>
		<tr >
			<td >CD45RB-PE IgG2a</td>
			<td >BD Pharminogen</td>
			<td >553101</td>
			<td></td>
		</tr>
		<tr >
			<td >Complete Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 10% FBS, 0.0004% &#946;-ME</td>
		</tr>
		<tr >
			<td >FACS tube + strainer</td>
			<td >BD Falcon</td>
			<td >352235</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Microscope Slides</td>
			<td >Fisherbrand</td>
			<td >12550A3</td>
			<td></td>
		</tr>
		<tr >
			<td >Heat-inactivated FBS</td>
			<td >Gemini</td>
			<td >100-106</td>
			<td></td>
		</tr>
		<tr >
			<td >Labeling Buffer</td>
			<td ></td>
			<td ></td>
			<td >1x PBS, 0.5% BSA, 2 mM EDTA</td>
		</tr>
		<tr >
			<td >Lysis Buffer</td>
			<td ></td>
			<td ></td>
			<td >0.08% NH4Cl, 0.1% KHCO3, 1 mM EDTA</td>
		</tr>
		<tr >
			<td >MoFlo XDP</td>
			<td >Beckman Coulter</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse CD4 T lymphocyte Enrichment Set - DM</td>
			<td >BD Biosciences</td>
			<td >558131</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2a-PE</td>
			<td >BD Pharminogen</td>
			<td >553457</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse IgG2b-FITC</td>
			<td >eBioscience</td>
			<td >11-4732</td>
			<td></td>
		</tr>
		<tr >
			<td >Pasteur pipet</td>
			<td >Fisherbrand</td>
			<td >13-678-20D</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin Solution, 100X</td>
			<td >Corning Cellgro</td>
			<td >30-002-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Petri Dish</td>
			<td >Fisherbrand</td>
			<td >875713</td>
			<td></td>
		</tr>
		<tr >
			<td >Pure Ethanol 200 Proof</td>
			<td >Decon Labs</td>
			<td >2705-HC</td>
			<td></td>
		</tr>
		<tr >
			<td >RPMI-1640</td>
			<td >Gibco</td>
			<td >11-875-093</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringe</td>
			<td >BD Biosciences</td>
			<td >309597</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypan blue</td>
			<td >Corning Cellgro</td>
			<td >25-900-CI</td>
			<td></td>
		</tr>
		<tr >
			<td >Wash Media</td>
			<td ></td>
			<td ></td>
			<td >RPMI-1640, 1% Pen/Strep, 0.0004% &#946;-ME</td>
		</tr>
	</tbody>
</table>

induction of murine intestinal inflammation by adoptive transfer of effector cd4+cd45rbhigh t cells into immunodeficient mice

有可用于研究人炎性肠疾病（IBD），每个都有其自己的优点和缺点的发病许多不同的动物模型。我们在这里描述通过过继转移的同源脾的CD4 + CD45RB 高 T细胞的成T和B细胞缺陷型受体小鼠启动的一个实验性结肠炎模型。在CD4 + CD45RB 高 T细胞群在很大程度上是由幼稚效应细胞能够诱导的慢性肠道炎症，非常类似于人类IBD的关键环节。这种方法可以被操纵来研究疾病发作和进展的方面。此外，它可以被用来研究先天，自适应的功能，以及调节免疫细胞群，和环境暴露的作用，也就是说 ，该微生物群，在肠道炎症。在这篇文章中，我们阐述了诱导结肠炎一步一步的协议的方法。这INC吕代的成功开发实验性结肠炎的研究目的这个小鼠模型所需要的关键技术环节的视频演示。

约10×10 6 CD4 + CD45RB 高Tg从成年C57BL / 6小鼠捐助10脾脏细胞是可靠的隔离。这个数目将根据患者的年龄的供体小鼠的应变和研究者的熟练程度而有所不同。当4×10 5只C57BL / 6的CD4 + CD45RB 高 T细胞转移到C57BL / 6 RAG1 - / -受体小鼠，疾病的临床症状出现围绕周5后饱食或更快，如果小鼠在遗传上易感更严重的疾病（ 图2，3）11,12。</sup...

Watch this Scientific Journal Video about Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice at JoVE.com

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Research

JoVE Journal

Immunology and Infection

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

实验转移和CTL过继转移免疫小鼠模型

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

科研

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流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

加速1型糖尿病感应在小鼠体内的过继转移致糖尿病的CD4 + T细胞

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

评估小鼠浆细胞树突状细胞在淋巴集结的开发利用造血祖细胞的过继转移

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

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分析<em&gt;小肠结肠炎耶尔森菌</em&gt;效应易位到宿主细胞中使用β-内酰胺酶效应融合体

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

从小鼠脾隔离和扩大不变的自然杀伤T细胞的优化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

在小鼠实验为细菌性肺炎的诱导和表型非侵入性和技术上非密集型方法

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

诱导麻痹及视觉系统损伤小鼠 T 细胞特异的视神经脊髓炎抗原水通道蛋白-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

立体定向移植人胶质瘤多形性移植小鼠细胞毒性免疫细胞模型的研究

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...