אינדוקציה של Murine דלקת מעיים על ידי העברת המאמצת של מפעיל CD4<sup> +</sup> CD45RB<sup> גבוה</sup> תאי T לעכברי immunodeficient
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
יש מודלים של בעלי חיים רבים ושונים זמינים ללימוד פתוגנזה של מחלות בבני אדם מעי דלקתיות (IBD), כל אחד עם יתרונות משלה וחסרונות. אנו מתארים כאן מודל קוליטיס ניסיוני שהוא ביוזמת העברת מאמצת של תאי טחול syngeneic CD4 + T CD45RB הגבוה לעכברי נמען חסרי T ו- B תא. אוכלוסיית תאי T מסוג CD4 + הגבוה CD45RB שמורכבת בעיקר של תאי מפעיל נאיביים היא מסוגלת גרימת דלקת מעיים כרונית, דומה מאוד להיבטים מרכזיים של IBD האנושי. שיטה זו ניתן להשפיע ללמוד היבטים של תחילת מחלה והתקדמות. בנוסף ניתן להשתמש בו כדי ללמוד את הפונקציה של מולדת, מסתגל, ואוכלוסיות תאים חיסוני רגולציה, ואת התפקיד של חשיפות סביבתיות, כלומר, החיידקים, בדלקת מעיים. במאמר זה אנו מדגימים את המתודולוגיה לגרימת דלקת המעי הגס עם פרוטוקול צעד-אחר-צעד. inc זהלרכב וידאו הדגמה של היבטים טכניים מפתח נדרשו לפתח מודל העכברי של קוליטיס הניסיוני למטרות מחקר בהצלחה.
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
כאן אנו מתארים פרוטוקול צעד-אחר-צעד גרימת דלקת במעי הגס בעכברים על ידי העברת מאמצת של תאי CD4 + CD45RB + T לעכברי immunodeficient. אנחנו השתמשנו טחולי C57BL / 6 תורם וRag1 syngeneic – / – עכברי נמען, אם כי זנים אחרים (למשל, BALB / ג, 129S6 / SvEv, סוכרת שאינן סובל מהשמנת יתר (NOD)) ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
עבודה זו נתמכה על ידי אמריקאי Gastroenterological Association (AGA) מחקר חוקרי פרס וקרוהן וקוליטיס קרן של אמריקה (CCFA) פיתוח קריירה פרס (לSZS), DK089692 F30 NIH NIDDK (לECS), ואוניברסיטת צפון מרכז קרוליינה לביולוגיה במערכת העיכול וDK34987 P30 מחלת גרנט (היסטולוגיה Core). מתקן UNC cytometry זרימת Core נתמך בחלקו על ידי NCI מרכז Core תמיכת גרנט (P30CA016086) למרכז לסרטן UNC Lineberger. אנו מודים ללוק B. Borst מState College אוניברסיטת צפון קרוליינה לרפואת וטרינרית על עזרתו עם ניתוח ואימונוהיסטוכימיה histopathological.
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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