AOM/DSS 结肠炎相关结肠肿瘤模型（文献参考）
氧化偶氮甲烷（AOM）化学启动、随后以 DSS 反复诱导结肠黏膜损伤与炎症所驱动的炎症相关结肠肿瘤发生范式，为结肠炎相关结直肠癌（CAC）研究中最常用的鼠类模型；其组织学与分子特征与人 CAC 高度相似，重复性好、成本低、易于实施；不同研究采用的品系与方案多样（综述所列方案即含 Balb/c、C57BL/6、C57BL/6J、Swiss Webster 等）。
Study design
以预先生成的 cDNA 为输入的 SYBR Green 实时定量 PCR 检测。每孔加入 10 ng cDNA，技术三重复；使用 PowerUp SYBR Green Master Mix 与 5 µM 正反向引物；按源方案循环条件扩增并读取 CT，以看家基因和参考实验组进行 ΔΔCT 分析。具体基因、引物和生物学分组按各研究设计确定。
Outcome measures
获得可供光学显微镜观察的 H&E 染色组织切片：细胞核蓝紫色、细胞质与结缔组织纤维粉红色，用于观察组织中细胞的整体结构与形态细节。本方案本身不产出病理评分数值，也不提供特定分子标志物的定量结果。
各样品目标序列的 CT 值；以看家基因归一化并以参考实验组计算 ΔΔCT/相对表达量。本方案不包含具体基因或疾病特异结论，输出判读依赖所选的引物、看家基因和实验分组。
每只动物的大体肿瘤数目、各肿瘤直径（卡尺测量）与肿瘤负荷（该动物全部肿瘤尺寸之和），配套照片与组织学分级输入；用于组间成瘤负担比较。
Experimental animals
- Age
- 8–12 周龄（AOM 给药开始时）
- Tissue
- colon（结肠，含远端结肠肿瘤）
- Tissue
- colon lamina propria mononuclear cells
- Age
- 8–12 周龄（AOM 给药开始时）
- Tissue
- colon（结肠，含远端结肠肿瘤）
- Age
- 8–12 周龄（AOM 给药开始时）
- Tissue
- colon（结肠，含远端结肠肿瘤）
- Age
- 未记载周龄，终点取材组织切片 HE 判读（离体）
- Tissue
- colon（结肠，含远端结肠肿瘤）
- Age
- 8–10 周龄开始 AOM/DSS 方案；终点约第 100 天肿瘤计数
- Sex
- Mixed
- Tissue
- colon（结肠，纵向剖开全长，大体肿瘤）
Experimental procedures
脱蜡（Dewaxing） 石蜡切片置于二甲苯中脱蜡 3 次，每次 00:03:00。 复水（Rehydration） 石蜡切片依次置于 100% 乙醇中 3 次、每次 00:03:00，再置于 95% 乙醇中 1 次、00:03:00，随后用水漂洗。 苏木素核染色（Hematoxylin staining） 石蜡切片以苏木素染液染色 00:00:20，随后在流水下冲洗 00:01:00。 分化与返蓝（Differentiation and bluing） 石蜡切片置于返蓝液中 00:01:00，随后在流水下冲洗 00:01:00；此步使细胞核呈现蓝色。 伊红染色（Eosin staining） 石蜡切片以伊红复染 00:00:10；此步使细胞质成分与结缔组织纤维呈现粉红色。 脱水与透明（Dehydration and Clearing） 石蜡切片依次置于 95% 乙醇中 00:02:00、100% 乙醇中 2 次、每次 00:02:00，随后置于二甲苯中 2 次、每次 00:02:00。 封片（Mounting） 石蜡切片以 Permount 封片介质加盖玻片封固。
qPCR 反应体系 **Option 2****: SYBR qPCR** qPCR 反应体系 Prepare SYBR Master mix (ul per well): 1. 5ul PowerUp SYBR Green Master Mix (#A25776, Applied Biosystem-ThermoFisher) 1. 1ul Primers (Forward and Reverse, diluted in Nuclease-free water at 5µM) qPCR 反应体系 Add 6ul of SYBR Master mix to each well with an automatic pipette (Multipette E3, Eppendorf) qPCR 反应体系 Add 4ul of cDNA per well (10ng cDNA) in technical triplicates to each well of LightCycler 480 Multiwell 384-plate (#4729749001, Roche Diagnostics) 热循环 Perform PCR using the following cycling conditions in a LightCycler® 480 System (Roche): 1. 50˚C for 2min 1. 95˚C for 2min 1. 95˚C for 15s + 60˚C for 1min (x40cycles) 数据分析 Using the LightCycler Software obtain the threshold cycles (CT) signals from each samples. 数据分析 Perform ΔΔCT-method to analyze the expression data using the endogenous control genes and the reference experimental group.
Procedure Open the mice using sterile forceps and scissors to carefully remove the entire colon and place it in a 10 cm Petri dish with 15 ml cold sterile PBS, which should be enough to cover the entire colon. Procedure Carefully hold the distal part of the colon with the help of forceps and then flush to remove feces with 5-10 ml cold sterile PBS using a 21 G needle attached to a 5 ml syringe. After flushing the feces, put the cleaned colon in a new Petri dish. Procedure Carefully open the colon longitudinally and rinse again with cold PBS to remove remaining feces. Procedure For the AOM/DSS experiment, count the macroscopic tumors in the longitudinally open colon. In our in-house bred mice with mostly C57BL/6 background, tumor development is observed in 100% of the WT animals. Tumor size (average diameter) is measured using a digital caliper and tumor load per mouse is calculated by summing up the sizes of all the tumors in a given mouse. After counting tumor numbers and load, longitudinally opened colons can be photographed (Figure 1) before fixing them as ”swiss-rolls” in 10% formalin solution and then embedding in paraffin. H&E stained paraffin sections (Figure 2) should be analyzed for tumor grading and immune cells infiltration by an experience pathologist.
Model identity
- Model name
- AOM/DSS 结肠炎相关结肠肿瘤模型（文献参考）
- Species
- mouse
- Taxonomy ID
- 10090
Biological characteristics
- Research questions
- 炎症驱动结直肠肿瘤发生的机制与病理序列；预防与治疗策略的临床前评价。
- Limitations
- 文献参考模型，不代表公司实际保有、供应或质检；属炎症驱动的结肠炎相关结直肠癌路径，不同于 Apc 基因显性点突变驱动的 Apc Min 遗传性多发肠肿瘤通路，也不覆盖散发性结直肠癌全谱；模型尚未标准化，不同研究的数据难以分析与比较；品系易感性差异显著（四品系同一方案对比：Balb/c 结肠腺癌发生率 100%、C57BL/6N 约 50%，C3H/HeN 与 DBA/2N 未见肿瘤，而炎症改变以 C3H/HeN 最明显），品系是决定成瘤与炎症结局的关键条件。
Experimental methods
Experimental protocols
- Hematoxylin and Eosin (H&E) staining for histopathology
Hematoxylin and eosin (H&E) staining of fixed, paraffin-embedded tissue sections (4 μm). The workflow covers dewaxing, rehydration, hematoxylin nuclear staining, bluing, eosin counterstaining, dehydration and clearing, and coverslipping, yielding blue-purple nuclei and pink cytoplasm and connective tissue fibers for light-microscopic assessment of cellular and tissue morphology.
- cDNA 模板的实时定量 PCR（qPCR，SYBR Green 分支）
This protocol adopts the SYBR Green branch of the protocols.io qPCR v1 record. The input is pre-generated cDNA (10 ng per well); RNA extraction and reverse transcription are not included. PowerUp SYBR Green Master Mix and forward/reverse primers are used on a LightCycler 480 with 50˚C for 2 min, 95˚C for 2 min, and 40 cycles of 95˚C for 15 s plus 60˚C for 1 min. CT values are read and analysed by the ΔΔCT method. No specific genes, primer sequences or disease-model readouts are asserted.
- Hematoxylin and Eosin (H&E) staining for histopathology
Hematoxylin and eosin (H&E) staining of fixed, paraffin-embedded tissue sections (4 μm). The workflow covers dewaxing, rehydration, hematoxylin nuclear staining, bluing, eosin counterstaining, dehydration and clearing, and coverslipping, yielding blue-purple nuclei and pink cytoplasm and connective tissue fibers for light-microscopic assessment of cellular and tissue morphology.
- Hematoxylin and Eosin (H&E) staining for histopathology
Hematoxylin and eosin (H&E) staining of fixed, paraffin-embedded tissue sections (4 μm). The workflow covers dewaxing, rehydration, hematoxylin nuclear staining, bluing, eosin counterstaining, dehydration and clearing, and coverslipping, yielding blue-purple nuclei and pink cytoplasm and connective tissue fibers for light-microscopic assessment of cellular and tissue morphology.
- Hematoxylin and Eosin (H&E) staining for histopathology
Hematoxylin and eosin (H&E) staining of fixed, paraffin-embedded tissue sections (4 μm). The workflow covers dewaxing, rehydration, hematoxylin nuclear staining, bluing, eosin counterstaining, dehydration and clearing, and coverslipping, yielding blue-purple nuclei and pink cytoplasm and connective tissue fibers for light-microscopic assessment of cellular and tissue morphology.
- 结肠大体肿瘤计数、卡尺直径测量与肿瘤负荷计算
Tumor-burden endpoint for the AOM/DSS colitis-associated cancer model: at the day-100 endpoint mice are euthanized by cervical dislocation, the entire colon is excised into 15 ml cold sterile PBS, flushed with 5-10 ml cold PBS through a 21 G needle, opened longitudinally and rinsed again; macroscopic tumors are counted one by one, each tumor's diameter is measured with a digital caliper, and tumor load per mouse is the summed size of all its tumors. Photographed colons are fixed in 10% formalin solution (Sigma-Aldrich HT-501128) for paraffin sections and histological grading. Applies to AOM/DSS models (predominantly C57BL/6 background) for comparing tumor number, size distribution and intervention effects; the adopted record reports 100% tumor take in its in-house C57BL/6-background wild-type mice at day 100.
Reagents and materials
- 组织学二甲苯（混合异构体）
脱蜡剂
- Ethanol
复水梯度溶剂（去除二甲苯）
- 95% 乙醇溶液
复水梯度溶剂
- 自来水（组织学染色用）
复水后漂洗用水（后续各流水冲洗共用）
- Epredia Signature Series 苏木素染液（7221）
细胞核染色
- Epredia Signature Series 返蓝液（7301）
返蓝（使细胞核转蓝）
- Epredia Signature Series 伊红 Y 染液（7111）
细胞质与结缔组织纤维复染
- Permount 组织学封片剂（Fisher SP15）
封片介质
- SYBR Green qPCR Master Mix
SYBR Green 实时 qPCR 扩增
- qPCR 正反向引物（5 µM）
SYBR Green qPCR 扩增引物
- 无核酸酶水（Nuclease-free water）
引物稀释
- Permount 组织学封片剂（Fisher SP15）
封片介质
- Epredia Signature Series 伊红 Y 染液（7111）
细胞质与结缔组织纤维复染
- Epredia Signature Series 返蓝液（7301）
返蓝（使细胞核转蓝）
- Epredia Signature Series 苏木素染液（7221）
细胞核染色
- 自来水（组织学染色用）
复水后漂洗用水（后续各流水冲洗共用）
- 95% 乙醇溶液
复水梯度溶剂
- Ethanol
复水梯度溶剂（去除二甲苯）
- 组织学二甲苯（混合异构体）
脱蜡剂
- 组织学二甲苯（混合异构体）
脱蜡剂
- Ethanol
复水梯度溶剂（去除二甲苯）
- 95% 乙醇溶液
复水梯度溶剂
- 自来水（组织学染色用）
复水后漂洗用水（后续各流水冲洗共用）
- Epredia Signature Series 苏木素染液（7221）
细胞核染色
- Epredia Signature Series 返蓝液（7301）
返蓝（使细胞核转蓝）
- Epredia Signature Series 伊红 Y 染液（7111）
细胞质与结缔组织纤维复染
- Permount 组织学封片剂（Fisher SP15）
封片介质
- 组织学二甲苯（混合异构体）
脱蜡剂
- Ethanol
复水梯度溶剂（去除二甲苯）
- 95% 乙醇溶液
复水梯度溶剂
- 自来水（组织学染色用）
复水后漂洗用水（后续各流水冲洗共用）
- Epredia Signature Series 苏木素染液（7221）
细胞核染色
- Epredia Signature Series 返蓝液（7301）
返蓝（使细胞核转蓝）
- Epredia Signature Series 伊红 Y 染液（7111）
细胞质与结缔组织纤维复染
- Permount 组织学封片剂（Fisher SP15）
封片介质
- PBS 磷酸盐缓冲液
结肠取材与冲洗介质
- 10% 中性缓冲福尔马林
大体计数后组织固定（备石蜡切片分级）
References
- Dzhalilova D, Zolotova N, Fokichev N, Makarova O. Murine models of colorectal cancer: the azoxymethane (AOM)/dextran sulfate sodium (DSS) model of colitis-associated cancer. PeerJ. 2023;11:e16159. DOI:10.7717/peerj.16159; PMID:37927787; PMCID:PMC10624171. 综述逐句支持：'For the study of colitis-associated cancer (CAC), the AOM/DSS model is predominantly used. This model is very similar in histological and molecular characteristics to the human CAC, and is highly reproducible, inexpensive, and easy to use. Despite its popularity, the AOM/DSS model is not standardized, which makes it difficult to analyze and compare data from different studies'；方案表列 Balb/C、C57BL/6、C57BL/6J、Swiss Webster 等品系；Suzuki 2006 四品系对比（Balb/c 100%、C57BL/6N 50%、C3H/HeN 与 DBA/2N 无肿瘤，炎症改变 C3H/HeN 最明显）；Apc Min 段落（Enu 诱导的 Apc 显性突变、多发肠新生物、杂合致病）。该综述提供范式地位、标准化状态与品系易感差异的背景证据。
- NCBI Taxonomy record 10090: Mus musculus (house mouse), rank species.
- Nathalia L. Oliveira; Samantha Gruenheid. Hematoxylin and Eosin (H&E) staining for histopathology. protocols.io, v1, 2026-07-31. DOI: 10.17504/protocols.io.kxygxr9jdg8j
- Epredia Signature Series Hematoxylin (7221), 产品信息页, Fisher Scientific
- Epredia Signature Series Bluing Reagent (7301), 产品信息页, Fisher Scientific
- Epredia Signature Series Eosin Y (7111), 产品信息页, Fisher Scientific
- Xylene, Fisherbrand HistoPrep (HC7001GAL), 产品信息页, Fisher Scientific
- Fisher Chemical Permount Mounting Medium (SP15 产品族), 产品信息页, Fisher Scientific
- Ethanol (CID 702, CAS 64-17-5), PubChem 化合物标准记录
- Miquel Vila. Gene expression analysis by quantitative Real-Time PCR (qPCR). protocols.io. https://doi.org/10.17504/protocols.io.ewov1oyz2lr2/v1
- Bustin SA, et al. MIQE précis: Practical implementation of minimum standard guidelines for quantitative real-time PCR experiments. BMC Molecular Biology 2010;11:74. PMID 20858237. doi:10.1186/1471-2199-11-74
- Gupta J, Nebreda AR. Induction of Colitis and Colitis-associated Colorectal Cancer (CAC). Bio-protocol 4(22): e1290 (2014). DOI: 10.21769/BioProtoc.1290
- ARRIVE 2.0 guidelines