【作者】 李璐；

【导师】 姜葵；

【作者基本信息】 天津医科大学 ， 内科学 消化系病， 2020， 硕士

【摘要】 目的结直肠癌(colorectal cancer,CRC)是世界范围内常见的十大恶性肿瘤之一,新近研究表明其发病率和死亡率位居第三位。特别是在中国地区,结直肠癌发病率和患病率逐渐增加。目前的研究认为CRC是一种多因素多步骤共同作用导致的疾病。目前已知的危险因素包括遗传易感性、年龄、性别、肥胖、生活方式以及环境因素等。约95%的肠道肿瘤是由腺瘤进展来的,但是机制尚未完全阐明。新近研究发现CRC患者的肠道菌群组成和功能与健康人明显不同,且致病菌更容易在肠腔定植。肠道微生物的直接致癌性及其衍生因子之间的相互作用激活致癌信号通路。从一定程度上说,CRC似乎是一种细菌相关性疾病。本研究旨在探索紊乱的肠道菌群在肠道腺瘤到腺癌的序列进展中的作用及机制。方法1、人体实验方法:我们收集了10名健康受试者和10名CRC患者的新鲜粪便按照标准流程制备粪菌液。所有受试者均经过严格的纳入和排除标准筛选,并签署了知情同意书。粪菌液分装后冻存于-80°C冰箱以备后续实验。2、动物实验方法:在接受3天的抗生素鸡尾酒的预处理后,4周龄的20只野生型C57BL/6J雌鼠随机分为2组,分别灌服健康受试者和CRC患者的粪菌液。4周龄的30只Apcmin/+雌鼠分为3组,分别灌服无菌的PBS溶液、健康受试者的粪菌液和CRC患者的粪菌液。在8周的实验周期里一共进行了16次的粪菌移植。处死前收集每只小鼠的新鲜粪便,分离小鼠的肝脏和脾脏、小肠组织及结肠组织。将小肠组织平均分为3段,靠近胃部段为近段,靠近回盲部段为远段。把各段肠道近端组织在液氮中快速冷冻后放置在-80°C保存,远端组织使用4%多聚甲醛固定。(1)记录各组小鼠的肠道腺瘤数量及分布,对小鼠的肠腺瘤组织进行石蜡切片行HE染色评价腺瘤病理分型;(2)对各组小鼠的肠道腺瘤石蜡切片行Ki-67免疫组织化学染色及原位末端转移酶标记技术评价肠道肿瘤细胞的增殖及凋亡情况;(3)行实时荧光定量聚合酶链反应(Real-time fluorescence quantitative polymerasechain reaction)实验检测肠道组织屏障功能及炎症因子的表达情况;(4)采用蛋白质免疫印迹法(Western blot)检测肠道肿瘤组织中Wnt信号通路相关分子蛋白的表达情况;(5)采用16S r DNA Amplicon测序检测不同处理条件下小鼠的肠道菌群差异。结果(1)与灌服无菌PBS溶液的小鼠和灌服健康受试者粪便样本的小鼠相比,灌服结直肠癌患者粪便样本的小鼠肠道腺瘤数目显著增加。(2)与灌服健康受试者粪便样本的小鼠相比,灌服结直肠癌患者粪便样本小鼠的腺瘤细胞Ki-67免疫组化染色阳性百分比明显增加,凋亡细胞染色百分比明显降低。(3)与灌服健康受试者粪便样本的小鼠相比,灌服结直肠癌患者粪便样本的小鼠肠道黏膜屏障功能相关紧密连接蛋白在m RNA水平的相对表达量下降,肠道炎症细胞因子在m RNA水平的相对表达量上升。(4)与灌服健康受试者粪便样本的小鼠相比,灌服结直肠癌患者粪便样本小鼠肠道腺瘤细胞β-catenin出现明显异位表达,cyclin D1阳性细胞比例明显增加。(5)与灌服健康受试者粪便样本的小鼠相比,灌服结直肠癌患者粪便样本的小鼠粪便中普沃氏菌属和螺杆菌属等条件致病菌丰度显著增加,梭菌属Ⅳ和XIVa以及罗氏菌属等产丁酸菌丰度减少。结论灌服结直肠癌患者粪便样本的Apcmin/+小鼠出现肠道黏膜屏障破坏和慢性低度炎症,Wnt/β-catenin相关信号通路活化,肠道菌群失衡从而促进肠腺瘤恶变。本研究为针对肠道菌群的CRC防治提供新的视角,肠道菌群的靶向干预似乎是治疗CRC的一种有前景的途径。更多还原

【Abstract】 ObjectiveColorectal cancer（CRC）,ranks the third in morbidity and mortality,is one of the most common malignant tumors in the world.The incidence and prevalence of colorectal cancer are increasing especially in China.Current studies suggest that CRC is a disease caused by multiple factors and multiple steps.The currently known risk factors of CRC including genetic predisposition and environmental factors such as age,gender,obesity,lifestyle.About 95%of intestinal tumors develop from adenomas,however its mechanisms have not been fully illuminated.Recent studies have found that the composition and function of intestinal microbiota in CRC patients are significantly different from that in healthy people.And pathogenic bacteria are more likely to colonize the intestinal cavity.The interaction between direct carcinogenicity of intestinal microbiota and its derived factors activates oncogenic signaling pathways.To some extent,CRC seems to be considered a bacteria-related disease.This study aimed to explore the role and mechanism of disordered intestinal microbiota in the progression of intestinal adenoma to adenocarcinoma.Methods1.Human experimental method:We collected fresh feces from 10 healthy subjects and 10 CRC patients to prepare faecal suspension according to standard procedures.All subjects who were screened by strict inclusion and exclusion criteria signed the informed consent.The faecal suspension was stored separately in-80°C refrigerator for subsequent experiments.2.Animal experimental method:After receiving three days of antibiotic cocktail pretreatment,the twenty conventional female C57BL/6J mice aged four weeks were randomly divided into two groups,one group was gavaged fecal samples from healthy controls,while the other group was gavaged fecal samples from CRC patients.The thirty Apc^min/+female mice aged 4 weeks were randomly divided into three groups,which were gavaged sterile PBS,fecal samples from healthy controls and fecal samples from CRC patients,respectively.The fecal microbiota transplantation（FMT）was repeated 16 times during the experimental period of 8 weeks.Fresh faeces from each mouse were collected before sacrifice.Then the liver,spleen,small intestine and colon tissues were isolated.The tissue of the small intestine was divided into three parts on average.The tissue near the stomach was defined as the proximal segment,and the tissue near the ileocecum was defined as the distal segment.The proximal tissue of each intestinal segment was snap frozen in liquid nitrogen and kept at-80°C later,and the distal tissue was fixed with 4%paraformaldehyde.（1）The number and distribution of intestinal adenomas of mice in each group were recorded.HE staining was performed on paraffin-embedded intestinal adenoma tissue to evaluate the pathological types.（2）Ki-67 immunohistochemical staining was performed on paraffin sections of intestinal adenomas in mice to evaluate the proliferation of intestinal tumor cells.Terminal deoxynucleotidyl transferase d UTP nick end labelling（TUNEL）was used to detect tumour cellular apoptosis.（3）Real time-PCR was performed to detect the intestinal barrier function and the expression of inflammatory factors.（4）Western blot was used to detect the expression of proteins related to the Wnt signaling pathway in intestinal tumor tissues.（5）16S r DNA Amplicon sequencing was used to detect the gut microbiota of mice under different treatment conditions.Results（1）The total number of adenomas in mice receiving fecal samples from CRC patients was increased compared with the PBS group,and the mice receiving fecal samples from healthy controls.（2）Compared with the mice receiving fecal samples from healthy controls,the positive percentage of Ki-67 immunohistochemical staining was significantly increased and the percentage of apoptotic cells was significantly decreased in the adenoma cells of mice receiving fecal samples from CRC patients.（3）The relative m RNA expression of tight junction protein associated with intestinal barrier function were decreased and the relative m RNA expression of intestinal inflammatory cytokines were increased in mice receiving fecal samples from CRC patients compared with mice receiving fecal samples from healthy controls.（4）Compared with mice receiving fecal samples from healthy controls,the mice receiving fecal samples from CRC patients showed significant heterotopic expression ofβ-catenin in intestinal adenoma cells,and the proportion of cyclin D1 positive cells increased significantly.（5）Compared with the mice which gavaged by the fecal samples from healthy controls,the abundance of Prevotella and Helicobacter increased in the mice which gavaged by the fecal samples from CRC patients.Meanwhile,the abundance of short-chain fatty acids（SCFAs）producing bacteria,such as ClostridiumⅣ,Clostridium XIVa,and Roseburia were significantly lower.ConclusionThe Apc^min/+mice which gavaged by the fecal samples from CRC patients showed intestinal barrier disruption,chronic low grade inflammation,activation of Wnt/β-catenin signaling pathways,and imbalance gut mcrobiota to promote the progression of intestinal adenoma.This study provided a new perspective for the prevention of CRC against gut mcrobiota.Targeted intervention against gut mcrobiota seems to be a promising way to treat CRC.更多还原