AOM/DSS结肠癌模型优化及其肠道菌群变化探究

Optimization of the azomethane oxide and dextran sodium sulfate model of colitis-associated colon cancer and changes in the intestinal microbiota

目的优化氧化偶氮甲烷(azoxymethane,AOM)联合葡聚糖硫酸钠(dextran sodium sulfate,DSS)造模结肠炎相关性结肠癌(colitis-associated colorectal cancer,CAC)方法并探究肠道菌群在CAC中的发病机制。方法通过AOM不同注射次数联合自由饮用DSS的方法建立A(AOM 1次注射)和B(AOM 2次注射)模型组,正常组采用腹腔注射生理盐水联合饮用纯净水,每组10只。造模结束后通过DAI评分、结肠长度、成瘤率及死亡率等指标综合评估,选择最佳的造模方案。然后对B模型组小鼠进行实验,取血清以ELISA法检测白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)及肿瘤标志物CA199、CEA、CA724含量;同时进行HE染色观察结肠病变;并对小鼠粪便进行16S rDNA高通量基因测序法分析,以探究CAC小鼠肠道菌群的变化。结果单次和加强AOM注射联合DSS均能诱导CAC小鼠模型。但与A模型组相比,B模型组小鼠结肠内增生物较大,排列紧密且形态大小较一致,成瘤率达到100%。与正常组相比,B模型组IL-6显著升高(P<0.05),TNF-α含量升高(P>0.05);肿瘤标志物除CA724外,CA199和CEA含量均明显升高(P<0.05);HE病理结肠内炎性细胞的浸润,并伴有管腔表面显示出高级别上皮内肿瘤样改变。菌群结果显示,与正常组相比,CAC小鼠肠道菌群物种多样性及丰度降低,疣微菌门和放线菌门增多(P<0.05),拟杆菌门和弯曲菌门减少(P<0.05)。阿克曼菌、普雷沃菌、瘤胃球菌、双歧杆菌等显著增多(P<0.05);罗氏菌属、Muribaculaceae、理研菌属、厌氧原体属等显著减少(P<0.05)。结论AOM 2次注射联合自由饮用1.5%(1.5 g/100 mL)DSS诱导的CAC模型小鼠结肠成瘤率高、肿瘤形态大小均一、死亡率低,可作为药效学实验评价的优选造模方案。多种肠道菌群紊乱或功能失调,造成的通透性增高,肠黏膜屏障功能破坏,进而诱发肠源性内毒素释放,导致持续的炎症反应或是诱发CAC发病的间接或直接原因。

Objective To optimize the method of combining azomethane oxide(AOM)and dextran sodium sulfate(DSS)to create a colitis-associated colon cancer(CAC)model,and to explore the pathogenesis of the intestinal flora in CAC.Methods Model groups A and B were established by one and two injections of AOM,respectively,combined with free drinking of DSS,and a normal control group was injected intraperitoneally with normal saline combined with purified water(n=10 mice per group).The better modeling scheme was selected by comprehensive evaluation of the disease activity index score,colon length,tumor rate,and mortality.Serum levels of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),and tumor markers CA199,CEA,and CA724 were detected by enzyme-linked immunosorbent assay.Colon lesions were evaluated by hematoxylin and eosin(HE)staining.Changes in the intestinal microbiota in CAC mice were detected by 16S rDNA high-throughput gene sequencing analysis of mouse feces.Results Both single and enhanced AOM injections combined with DSS induced CAC mice;however,colon growths were larger,more closely arranged,and their morphological size was more consistent in group B compared with group A,with a tumor-formation rate of 100%.IL-6 levels were increased in the model group compared with the normal group(P<0.05).TNF-αlevels were increased in the model group compared with the normal group(P>0.05).The CA199 and CEA levels were also significantly increased(P<0.05),but CA724 levels were not.Infiltration of inflammatory cells in the colon detected by HE pathology was accompanied by high-grade intraepithelial tumor-like changes on the surface of the lumen.The diversity and abundance of intestinal bacteria were decreased in CAC mice compared with normal mice:phyla Verrucomicrobiota and Actinobacteriota were significantly increased(P<0.05),Bacteroidota and Campilobacterota were significantly decreased(P<0.05).Akkermansia,Prevotellaceae,Ruminococcus,and Bifidobacterium were significantly increased(P<0.05),and Roseburia,Rikenellaceae_RC9_gut_group,Anaeroplasma,and Muribaculaceae were significantly decreased(P<0.05).Conclusions Two injections of AOM combined with 1.5%(1.5 g/100 mL)DSS induced CAC model mice with a high colon-tumorigenesis rate,uniform tumor morphology,and low mortality,and may thus be the preferred modeling scheme for pharmacodynamic experiments.Disorders or dysfunction of the intestinal flora may lead to increased permeability,loss of intestinal mucosal barrier function,and the release of enterogenic endotoxins,Resultsing in a sustained inflammatory response,as an indirect or direct cause of CAC pathogenesis.