Efektör CD4 Evlatlık transferi ile Kemirgen Bağırsak Enflamasyon indüksiyonu<sup> +</sup> CD45RB<sup> Yüksek</sup> Bağışık yetmezliği olan farelere içine T Hücreleri
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
İnsan inflamatuar barsak hastalıklarının (IBD), kendi avantajları ve dezavantajları her patogenezi çalışmak için birçok farklı hayvan modelleri vardır. Burada T ve B hücresi eksik alıcı farelere singeneik dalak CD4 + CD45RB yüksek T hücrelerinin adoptif nakli ile başlatılır deneysel kolit modelini açıklar. Büyük ölçüde naif efektör hücrelerin oluşur CD4 + T hücre CD45RB yüksek popülasyonu yakından insan IBD'nin kilit yönlerini benzeyen, kronik bağırsak inflamasyonu uyarma yeteneğine sahiptir. Bu yöntem, hastalığın başlangıcında ve ilerlemesinde yönlerini incelemek için manipüle edilebilir. Ayrıca bağırsak iltihabı yani doğuştan gelen, adaptif fonksiyonlarını ve düzenleyici immün hücre popülasyonları ve çevresel maruziyet rolü, Mikrobiyota, incelemek için kullanılabilir. Bu yazıda, bir adım-adım protokolü ile Kolit için bir metodoloji göstermektedir. Bu incidarecisi başarıyla araştırma amaçlı deneysel kolit bu fare modeli geliştirilmesi için gerekli anahtar teknik yönlerini bir video gösterisi.
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
Burada bağışıklık yetersizliği olan farelere, CD4 + CD45RB + T hücrelerinin adoptif nakli ile farelerde kolon inflamasyonu yol açan bir adım adım protokol açıklar. Alıcı fareler başka soybaşları rağmen (örneğin, BALB / c, 129S6 / SvEv, obez olmayan diyabetik (NOD)) immün yetmezlik ve genetik modelleri (örneğin, SCID, Rag2 – / – Bu, C57BL / 6 verici dalak ve genetik olarak özdeş Rag1 kullanılan – / -) da 4,14-16…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Bu çalışma, Amerikan Gastroenteroloji Derneği (AGA) Araştırma Alimler Ödülü ve Crohn ve Amerika (CCFA) (ECS) (SZS için) Kariyer Geliştirme Ödülü NIH NIDDK F30 DK089692 Kolit Vakfı ve Gastrointestinal Biyoloji Kuzey Carolina Merkezi'nin Üniversitesi tarafından desteklenen ve Hastalık Hibe P30 DK34987 (Histoloji Çekirdek). UNC Sitometrisi Çekirdek Tesisi UNC Lineberger Kapsamlı Kanser Merkezi'nde bir NCI Merkezi Çekirdek Desteği Grant (P30CA016086) tarafından kısmen desteklenmektedir. Biz histopatolojik analiz ve immunohistokimyasal ile yaptığı yardım için Veteriner North Carolina State University College Luke B. Borst teşekkür ederim.
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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