エフェクターCD4の養子移入によるマウス腸の炎症の誘導<sup> +</sup> CD45RB<sup>高い</sup>免疫不全マウスへのT細胞
Summary
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.
Abstract
ヒト炎症性腸疾患（IBD）、それぞれに長所と短所の病因を研究するために利用可能な多くの異なる動物モデルがある。ここではTおよびB細胞欠損レシピエントマウスに同系の脾臓CD4 + CD45RB high T細胞の養子移入によって開始される実験的大腸炎モデルを記述する。主にナイーブ、エフェクター細胞からなるCD4 + CD45RB 高 T細胞集団は、密接に、ヒトIBDの重要な側面に似た、慢性的腸炎を誘導することができる。この方法は、疾患の発症および進行の側面を研究するために操作することができる。さらに、それは腸の炎症、すなわち 、先天性、適応性の機能、及び調節免疫細胞集団、および環境曝露の役割は、微生物を研究するために使用することができる。この記事では、ステップバイステップのプロトコルで大腸炎を誘発するための方法論を示している。このINCludes成功裏に研究目的のために実験的大腸炎のこのマウスモデルを開発するために必要なキー技術的な側面のデモビデオ。
Introduction
The inflammatory bowel diseases (IBD) Crohn’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 naïve 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.
Protocol
Representative Results
Discussion
ここでは、免疫不全マウスにCD4 + CD45RB + T細胞の養子移入によってマウスでの大腸の炎症を誘導するステップバイステップのプロトコルを記述します。 – / –レシピエントマウス、他の系統であるが（ 例えば 、BALB / C、129S6 / SvEv、非肥満糖尿病（NOD））及び免疫不全の遺伝的モデル（ 例えば 、SCID、 が、Rag2我々はC57BL / 6ドナー脾臓と同系RAG1を</em…
Disclosures
The authors have nothing to disclose.
Acknowledgements
この作品はアメリカの消化器協会（AGA）研究学者賞とクローン病とアメリカの大腸炎財団（SZSへ）（CCFA）キャリア開発賞、（ECS）は、NIH NIDDK F30 DK089692、胃腸生物学ノースカロライナ大学のセンターによってサポートされていましたと疾病グラントのP30のDK34987（組織学コア）。 UNCフローサイトメトリーコアファシリティは、UNCラインバーガー総合がんセンターのNCIセンターコアサポートグラント（P30CA016086）によって部分的にサポートされています。私たちは、病理組織学的分析および免疫組織化学との彼の助けを獣医のノースカロライナ州立大学からルークB.ボーストに感謝。
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| 10x PBS | Gibco | 14200075 | |
| 12x75mm round-bottom tube | Falcon | 352052 | |
| 15 ml conical | Corning | 430790 | |
| 26g x 3/8 Needle | BD Biosciences | 305110 | |
| 50 ml conical | Corning | 430828 | |
| 70 um Cell Strainer | Fisherbrand | 22363548 | |
| BD IMagnet | BD Biosciences | 552311 | |
| β-mercaptoethanol | Thermo Scientific | 35602 | |
| CD4-FITC IgG2b | eBioscience | 11-0041 | |
| CD45RB-PE IgG2a | BD Pharminogen | 553101 | |
| Complete Media | RPMI-1640, 1% Pen/Strep, 10% FBS, 0.0004% β-ME | ||
| FACS tube + strainer | BD Falcon | 352235 | |
| Glass Microscope Slides | Fisherbrand | 12550A3 | |
| Heat-inactivated FBS | Gemini | 100-106 | |
| Labeling Buffer | 1x PBS, 0.5% BSA, 2 mM EDTA | ||
| Lysis Buffer | 0.08% NH4Cl, 0.1% KHCO3, 1 mM EDTA | ||
| MoFlo XDP | Beckman Coulter | ||
| Mouse CD4 T lymphocyte Enrichment Set – DM | BD Biosciences | 558131 | |
| Mouse IgG2a-PE | BD Pharminogen | 553457 | |
| Mouse IgG2b-FITC | eBioscience | 11-4732 | |
| Pasteur pipet | Fisherbrand | 13-678-20D | |
| Penicillin-Streptomycin Solution, 100X | Corning Cellgro | 30-002-CI | |
| Petri Dish | Fisherbrand | 875713 | |
| Pure Ethanol 200 Proof | Decon Labs | 2705-HC | |
| RPMI-1640 | Gibco | 11-875-093 | |
| Syringe | BD Biosciences | 309597 | |
| Trypan blue | Corning Cellgro | 25-900-CI | |
| Wash Media | RPMI-1640, 1% Pen/Strep, 0.0004% β-ME |
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