تحريض الفئران المعوية التهاب عن طريق نقل بالتبني من المستجيب CD4<sup> +</sup> CD45RB<sup> عالية</sup> خلايا T في الفئران العوز المناعي
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
هناك العديد من النماذج الحيوانية المختلفة المتاحة لدراسة المرضية للأمراض البشرية التهابات الأمعاء (IBD)، ولكل منها مزاياه وعيوبه. وصفنا هنا نموذج التهاب القولون التجريبية التي بدأها نقل بالتبني من خلايا الطحال مسانج CD4 + T CD45RB عالية إلى T والخلايا B الفئران المتلقي ناقصة. السكان خلية عالية T CD4 + CD45RB التي تتكون في معظمها من خلايا المستجيب ساذجة غير قادرة على إحداث التهاب الأمعاء المزمن، تشبه عن كثب الجوانب الرئيسية لIBD البشري. هذه الطريقة يمكن التلاعب بها لدراسة جوانب من بداية ظهور أعراض المرض والتقدم. بالإضافة إلى ذلك فإنه يمكن استخدامها لدراسة وظيفة الفطري، على التكيف، والسكان الخلايا المناعية التنظيمي، ودور التعرض للعوامل البيئية، أي الجراثيم، في التهاب الأمعاء. في هذه المادة ونحن توضيح المنهجية لإحداث التهاب القولون مع بروتوكول خطوة بخطوة. هذا المؤتمر الوطني العراقيLUDES مظاهرة الفيديو من الجوانب التقنية الأساسية اللازمة لتطوير هذا نموذج الفئران من التهاب القولون التجريبي لأغراض البحث بنجاح.
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
نحن هنا وصف بروتوكول خطوة بخطوة إحداث التهاب القولون في الفئران عن طريق نقل بالتبني من خلايا CD4 + T + CD45RB في الفئران العوز المناعي. كنا الطحال C57BL / 6 المانحة وRag1 مسانج – / – الفئران المتلقي، على الرغم من سلالات أخرى (على سبيل المثال، BALB / ج، 129S6…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
وأيد هذا العمل من قبل الجمعية الأمريكية الجهاز الهضمي (AGA) بحوث جائزة العلماء وكرون والتهاب القولون مؤسسة الأمريكية (CCFA) جائزة التطوير المهني (لSZS)، NIH NIDDK F30 DK089692 (لECS)، ومركز جامعة نورث كارولينا للبيولوجيا الجهاز الهضمي ومرض غرانت P30 DK34987 (علم الأنسجة الأساسية). ويدعم مرفق التدفق الخلوي UNC الأساسية في جزء منه من قبل مركز NCI الأساسية دعم جرانت (P30CA016086) إلى مركز UNC Lineberger الشامل للسرطان. ونحن نشكر لوقا B. بورست من ولاية كارولينا الشمالية جامعة كلية للطب البيطري لمساعدته مع تحليل الأنسجة والمناعية.
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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