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

作　　者：王敦方 朱琳 冯雪[1] 张彩娟 刘海帆 刘雅清 刘滨 刘丽 杨伟鹏[1] WANG Dunfang;ZHU Lin;FENG Xue;ZHANG Caijuan;LIU Haifan;LIU Yaqing;LIU Bin;LIU Li;YANG Weipeng(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Heilongjiang University of Chinese Medicine,Haerbin 150040,China)

机构地区：[1]中国中医科学院中药研究所,北京100700 [2]黑龙江中医药大学,哈尔滨150040

出　　处：《中国实验动物学报》2024年第2期151-160,共10页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家自然科学基金(82074328,81473592);中国中医科学院科技创新工程项目(CI2021A04604);中国中医科学院中药研究所自选课题-人才引进项目(ZXKT20033)。

摘　　要：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

关 键 词：结直肠癌 动物模型 肠黏膜屏障 炎症 肠道菌群 

分 类 号：Q95-33[生物学—动物学]