大鼠食管癌变及其化学预防研究

发布时间：2018-03-13 06:51

本文选题：食管癌　切入点：干细胞　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

【摘要】：目前,食管癌占全世界范围内癌症死亡率的第六位,在我国位于癌症死亡率的第四位。食管癌在全世界不同地区和人种间的发病率有着很大的不同,因此环境因素与遗传因素均与食管癌的发病相关。食管癌主要包括食管鳞状细胞癌和食管腺癌两大病理类型,其中食管鳞状细胞癌约占食管恶性肿瘤的90%,我国中部地区及中亚是食管鳞癌的主要流行地区。在我国食管癌病因学研究领域,上世纪七八十年代通过对河南省林州市食管癌高发现场深入的病因学研究明确了亚硝胺类物质、霉变食物、微量元素缺乏等是我国食管癌的重要化学病因。这其中,在林县食管癌高发现场分离得到的甲基苄基亚硝胺是目前已知最强的诱导大鼠食管癌的化学致癌物,并且其诱导的大鼠食管癌病理形态与人食管鳞状细胞癌的发生时序相平行,因此,甲基苄基亚硝胺主要被用来构建大鼠食管癌动物模型。该动物模型对研究食管癌的发生、发展机制及化学预防有着重要意义。对于上皮来源实体瘤的研究如实体瘤内部不同细胞体外克隆形成能力的差异,使得人们对肿瘤异质性有了更深入的认识。肿瘤干细胞理论的提出为解释肿瘤细胞的异质性提供了一定的理论依据。目前,食管鳞癌干细胞标志物的研究仍处于探索阶段,特异程度较高的标志物依然有待进一步寻找。二甲双胍是目前应用最广泛的2型糖尿病治疗药物,近期一些流行病学研究显示二甲双胍可以降低多种实体瘤的发生,这其中二甲双胍降低食管癌发生的流行病学研究也偶有报道,但二甲双胍降低食管癌发生发展的分子机制却未见明确报道。本研究中,我们首先构建了两种NMBzA给药途径诱导的大鼠食管癌动物模型并优化动物模型,得到了从增生、不典型增生到癌阶段的动态病理标本。免疫组化染色分析了目前被证实的大鼠食管干细胞标志物及人食管鳞癌干细胞标志物等在大鼠食管癌发生发展过程中的动态变化情况。其次,我们利用NMBzA皮下给药模型进行了二甲双胍预防食管鳞癌发生的动物模型研究,证实了二甲双狐能够有效抑制NMBzA诱导的大鼠食管癌及其癌前病变的发生与发展,并揭示了 AMPK/mTOR信号转导通路是二甲双胍抑制食管癌发生的主要作用靶点。最后,通过体外细胞实验进一步证实二甲双胍可以抑制食管上皮永生化细胞与食管癌细胞的增殖能力,使上述细胞的细胞周期阻滞在G0/G1期,并促进细胞凋亡的发生,上述现象与二甲双胍抑制食管癌细胞Bcl-2及cyclinD1的表达相关。此外,食管癌细胞的迁移运动能力与平板克隆形成能力及成球性生长能力也显著受到抑制,这与二甲双胍影响食管癌细胞中AMPK/mTOR、PI3K/Akt及Stat3信号转导通路和降低干性相关基因表达相关。综上所述,我们的研究成功构建了 NMBzA诱导的大鼠食管癌动物模型,为大鼠食管癌发生发展过程中各重要基因的突变与表达量的改变以及研究食管鳞癌干细胞的起源问题提供了标本来源。通过体内、体外实验证实了二甲双胍预防食管癌发生发展的分子机制,为二甲双胍在临床中预防及治疗食管癌癌前病变提供一定的理论基础。
[Abstract]:At present, esophageal cancer accounted for worldwide cancer mortality in China in sixth, cancer mortality fourth. The incidence of esophageal cancer in the world in different regions and races are very different, so the incidence of environmental and genetic factors were associated with esophageal cancer esophageal cancer mainly includes two. Pathological types of esophageal squamous cell carcinoma and adenocarcinoma of the esophagus, and esophageal squamous cell carcinoma accounts for about 90% of malignant tumor of esophagus, the central region of China and Central Asia is the major epidemic areas of esophageal cancer. In the field of study on etiology of esophageal cancer in China, the last century in 70s and 80s by the Linzhou city of Henan Province, a high incidence of esophageal cancer etiology research field further clear the nitrosamines, moldy food, lack of trace elements are important chemical etiology of esophageal cancer in China. Among these, the separated methyl in Lin County, a high risk area of esophageal cancer Benzyl nitrosamine induced esophageal cancer in rats is currently the strongest known chemical carcinogens, and the timing of rats induced by esophageal cancer and pathological morphology of human esophageal squamous cell carcinoma are parallel, therefore, METHYLBENZYLNITROSAMINE mainly is used to construct esophageal carcinoma animal model rats. The animal model study on esophageal cancer, has important significance and development mechanism of chemoprevention. For solid tumors of epithelial origin such as differences in tumor cells in vitro in different colony forming ability, which make people have a deeper understanding of tumor heterogeneity. The cell theory provides a theoretical basis for the interpretation of tumor heterogeneity the tumor stem cells. At present, esophageal squamous cell carcinoma stem cell marker research is still in the exploratory stage, the higher degree of specific markers remains to be further search. Metformin is the most Type 2 diabetes wide drug treatment, some recent epidemiological studies suggest that metformin may reduce the occurrence of a variety of solid tumors, which metformin reduces epidemiological study on esophageal cancer have also been reported, but the molecular mechanism of metformin reduces the development of esophageal cancer has not been clearly reported. In this study, and optimize the animal model of esophageal cancer in rats the animal model we first constructed two NMBzA administration induced, obtained from hyperplasia, dysplasia and carcinoma specimens dynamic pathological stage. Immunohistochemical staining analysis confirmed the rat esophageal stem cell markers and human esophageal squamous cell carcinoma stem cell markers have dynamic changes in the development process in esophageal carcinoma rats. Secondly, we use the NMBzA model by subcutaneous administration of metformin on animal model of esophageal carcinoma, syndrome The two armor double fox to the occurrence and development of esophageal carcinoma rats effectively inhibit NMBzA induced and precancerous lesions, and reveals the AMPK/mTOR signal transduction pathway is the main effect of metformin inhibited esophageal cancer target. Finally, through in vitro experiments further confirmed that two metformin can inhibit the proliferation ability of human immortalized esophageal epithelial cells with esophageal cancer cells, block the cell cycle of the cells in the G0/G1 phase, and promotes cell apoptosis, the expression of the above phenomenon with metformin inhibits Bcl-2 and cyclinD1 esophageal cancer cells. In addition, esophageal cancer cell migration and colony forming ability and Ballability growth ability also significantly inhibited. The effects of metformin and AMPK/mTOR in esophageal carcinoma cells, PI3K/Akt and Stat3 signal transduction pathway and reduce the stemness related genes expression. In summary, I Our study successfully constructed the animal model of NMBzA induced rat esophageal cancer, esophageal cancer rat mutated each important gene during the development and expression and the origin of esophageal squamous cell carcinoma stem cells provide a source of specimen. In vivo, in vitro experiments confirmed the molecular mechanism of the occurrence and development of esophageal and metformin for the prevention of cancer, and provide a theoretical basis for the treatment of esophageal precancerous lesions of metformin in clinic.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.1

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