次级胆汁酸诱导Apcmin/+小鼠肠腺瘤癌变及其对肠道菌群的影响

摘要

目的 观察并分析次级胆汁酸诱导A pcmin/+小鼠肠道腺瘤癌变及其对肠道菌群的影响.方法 将20只4周龄Apcmin/+小鼠均分为A pcmin/+对照组、A pcmin/+脱氧胆酸组,将20只4周龄野生型C57BL/6J小鼠均分为野生型对照组、野生型脱氧胆酸组.对照组常规饮水,脱氧胆酸组饮用水中含0.2％脱氧胆酸.收集给药0周及12周A pcmin/+小鼠粪便,焦磷酸测序法分析肠道菌群变化.12周后处死,观察各组小鼠肠道腺瘤数目、大小及分布.采用HE染色评价腺瘤病理类型,采用免疫组织化学染色法检测增殖细胞核抗原(PCNA),采用脱氧核苷酸末端转移酶介导的dUTP缺口末端标记法(TUNEL)检测细胞凋亡.两组间计量资料的比较采用独立样本t检验.结果 所有野生型小鼠肠道均未出现肿瘤.Apcmin/+脱氧胆酸组小鼠肠道腺瘤总数较A pcmin/+对照组明显增加(57.00±3.07比21.50±4.69,t=20.03,P＜0.01),以最大径为1～2 mm的腺瘤增加最为显著(30.62±7.73比7.75±4.59,t=8.04,P＜0.05),腺瘤癌变率较Apcmin/+对照组也明显上升.Apcmin+脱氧胆酸组小鼠肠道肿瘤PCNA阳性细胞百分比较Apcmin/+对照组显著增加[(90.17±2.14)％比(41.97±4.26)％,t=31.97,P＜0.01)],肿瘤细胞凋亡百分比显著减少[(1.40±1.12)％比(7.50±0.65)％,t=14.90,P＜0.01].Apcmin/+脱氧胆酸组小鼠肠道菌群多样性明显降低,在菌门水平上厚壁菌门与拟杆菌门比值(0.586 7±0.148 4)较其对照组(0.387 3±0.013 6)明显升高(t=2.36,P＜0.05);在菌属及菌种水平上Apcmin/+脱氧胆酸组致病菌数量增多,益生菌数量明显降低.结论 脱氧胆酸能诱导Apcmin/+小鼠肠道菌群失衡,并可通过增加肠道肿瘤细胞增殖,抑制细胞凋亡促使肠道腺瘤发展成为肠癌.%Objective To investigate secondary bile acid induced canceration process of intestinal adenoma and effects on intestinal microflora in Apcmin/+ mice.Methods Forty four-week-old mice (20 Apcmin/+mice and 20 wild-type C57BL/6J mice) were divided into four groups:wild-type control group (regular drinking water),wild-type deoxycholic acid (DOC) group (with 0.2 ％ DOC in drinking water),Apcmin/+ control group and Apcmin/+ DOC group.Fecal pellets of Apcmin/+ mice were collected at 0 week and 12 week after administration.The changes of intestinal microflora were analyzed by pyrosequencing.All mice were sacrificed after 12 weeks.The number,size and location of intestinal adenoma were observed.The pathological type of adenoma was evaluated after hematcxylin-eosin (HE) staining.Proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry.Cell apoptosis was determined by in situ terminal deoxynucleotidyl transferase mediated dUTP nick end labeling technique (TUNEL).Independent t test was used for the quantitative data comparison between two groups.Results No intestinal tumors were found in the wild-type mice.The total number of intestinal adenoma of Apcmin/+ DOC group significantly increased,compared with that of Apcmin/+ control group (57.00 ± 3.07 vs 21.50± 4.69,t=20.03,P＜0.01),the increase of the adenoma with maximum diameter between 1 to 2 mm was most significant (30.62± 7.73 vs 7.75 ± 4.59,t =8.04,P＜ 0.05),the rate of adenoma canceration also significantly increased compared with that of Apcmin/+ control group.The percentage of PCNA positive cells significantly increased compared with that of Apcmin/+ control group ((90.17 ± 2.14) ％ vs (41.97 ± 4.26) ％,t=31.97,P＜0.01).The percentage of cell apoptosis significantly declined ((1.40± 1.12) ％ vs (7.50 ± 0.65)％,t =14.90,P＜ 0.01).The diversity of intestinal flora of Apcmin/+ DOC group significantly decreased.The ratio of Firmicutes and Bacteroidetes significantly increased compared with control group (0.586 7±0.148 4 vs 0.387 3±0.013 6,t=2.36,P＜0.05).The number of pathogenic bacteria increased in Apcmin/+ DOC group and probiotics significantly decreased.Conclusion DOC can induce intestinal flora imbalance in Apcmin/+ mice and promote intestinal adenoma into adenocarcinoma through increasing tumor cell proliferation and inhibiting cell apoptosis.