Modeling Colitis-Associated Cancer med Azoxymethane (AOM) og dextransulfat Natrium (DSS)
Summary
Vi viser en protokol, hvor administration af genotoksisk stof azoxymethane (AOM) efterfulgt af tre cyklusser af det pro-inflammatoriske middel dextransulfat-natrium (DSS) hurtigt og konsekvent frembringer colontumorer hos mus med morfologiske og molekylære lighed med dem, der ses i human colitis -associeret cancer.
Abstract
Personer med inflammatorisk tarmsygdom (IBD), såsom Crohns sygdom (CD) eller colitis ulcerosa (UC) er i øget risiko for at udvikle kolorektal cancer (CRC) over raske personer. Denne risiko er proportional med varigheden og omfanget af sygdom, med en kumulativ incidens så høj som 30% hos individer med langvarige UC med udbredt colon engagement. 1 Colonic dysplasi i IBD og colitis associeret cancer (CAC) menes at udvikle sig som et resultat af gentagne cykler af epitelial-celle skade og reparation, mens disse celler bades i en kronisk inflammatorisk cytokin miljø. 2 Mens spontane og colitis-associerede cancere deler kvalitet er adenocarcinomer er sekvensen af de underliggende molekylære begivenheder menes at være anderledes. 3. Denne skelnen argumenterer behovet for specifikke dyremodeller for CAC.
Adskillige musemodeller øjeblikket findes for studiet af CAC. Dextransulfat Sodium (DSS), et middel med direkte toksiske virkninger på tyktarmsepitel, kan indgives i drikkevand til mus i flere cyklusser for at skabe en kronisk inflammatorisk tilstand. Med tilstrækkelig varighed, vil nogle af disse mus udvikler tumorer. Fire tumorudviklingen er fremskyndes i denne model hvis den gives i en pro-kræftfremkaldende indstilling. Disse omfatter mus med genetiske mutationer i tumorigenese pathways (APC, p53, MSH2), såvel som mus forbehandlet med genotoksiske midler (azoxymethane [AOM], 1,2-dimethylhydrazin [DMH]). Fem
Kombinationen af DSS med AOM som en model for colitis associeret cancer har vundet popularitet for sin reproducerbarhed, styrke, lav pris, og brugervenlighed. Selv om de har en fælles mekanisme, der AOM vist sig at være mere potente og stabile i opløsning end DMH. Mens tumorudvikling hos andre modeller kræver almindeligvis flere måneder, mus injiceret med AOM og efterfølgende behandlet med DSS udvikle passende tumorer i ens lidt som 7-10 uger. 6, 7 Endelig AOM og DSS kan administreres til mus af alle genetiske baggrund (knock out, transgene osv.) uden krydsning til en specifik tumorigen stamme. Her viser vi en protokol for inflammation drevet colon tumorigenese i mus under anvendelse af en enkelt injektion af AOM efterfulgt af tre syv-dages cyklusser af DSS over en 10 ugers periode. Denne model fremkalder tumorer med histologiske og molekylære forandringer ligger tæt op ad dem, der forekommer i human CAC og giver en meget værdifuld model til undersøgelse af onkogenese og chemoprevention i denne sygdom. 8
Protocol
Discussion
Behandling af mus med AOM og DSS hurtigt og effektivt modeller human colitis-associeret cancer. Hypoteser vedrørende arvelige faktorer, colitis-associerede cancere er let studeret med genetisk modificerede mus. 13, 16 Alternativt kan virkningen af farmakologiske mål i colitis-associeret cancer blive undersøgt ved anvendelse af vild-type mus.
Mens denne model er stærkt værdsat af dem interesseret i studiet af colontumor udvikling i fastsættelsen af inflammation, findes …
Divulgations
The authors have nothing to disclose.
Acknowledgements
Dette arbejde blev finansieret delvist af DK089016 og L30 RR030244 (MAC), CA153036 (AS), og P30-DK52574 (til Washington University Digestive Diseases Research Core). AIT var en Howard Hughes Medical Institute Medical Research Training Fellow.
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| C57BL/6J Mice | Jackson Laboratory | 000664 | |
| Azoxymethane (AOM) | Sigma Aldrich | A5486-100MG | Stock solution: dilute to 10 mg/ml in distilled water to be kept at -20 °C as 0.5 – 1 ml aliquots.
Working solution: dilute stock to 1 mg/ml in isotonic (0.9%) saline |
| Dextran Sulfate Sodium (DSS) | TdB Consultancy | DB001 | MW 40 kDa (36-50 kDa preparations from other sources are acceptable; The same lot should be used for a single experiment)6 |
| Coloview miniendoscopic system | Karl Storz | Multiple | See Becker et al. for detailed explanation of equipment and setup.11 |
| TPP Rapid FILTERMAX 500 ml Bottle-Filter, 0.22 μm PES | Midwest Scientific | TP99500 | Any standard tissue culture filter is acceptable |
| Ethyl Alcohol 200 Proof ASC/USP | Pharmaco-AAPER (or other) | 11ACS200 | Dilute to 70% in distilled water |
| Isoflurane, USP | Butler Animal Health Supply | 4029405 | Place mouse in glass jar with gauze or a small cloth soaked in anesthetic |
| 18G Straight Gavage Needle | Braintree Scientific | N-008 | |
| Phosphate Buffered Saline (PBS) | Sigma Aldrich | P5493 | Dilute to 1X (0.01 M) in distilled water |
| Cold Tray (Tissue Tek II Cold Plate) | Fisher Scientific | NC9491941 | Store at -20 °C |
| ImageJ Software | NIH (free download) | http://rsbweb.nih.gov/ij/ | |
| Formaldehyde (37%) | Fisher Scientific | F79-500 | Dilute to 10% in PBS |
| BD Bacto Agar | Fisher Scientific | DF0140-01-0 | Use hotplate to create 2% solution in distilled water |
| Miltex Eye Dressing Forceps | MedPlus Inc. | 18-780 | |
| Miltex Eye Scissors | MedPlus Inc. | 18-1430 | Curved points prevent damage to colon during opening. |
| Alcian Blue 8GX (powder) | Sigma Aldrich | A5268 | Add 1 g powder to 100 ml 3% acetic acid (3 ml glacial acetic acid + 97 ml distilled water) |
| 1 mL Tuberculin syringe with attached 26 G x 3/8 in intradermal bevel needle | BD | 305946 | For injection of AOM |
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