具核梭杆菌促进大肠癌发生的相关性研究
发布时间：2018-08-14 16:27
【摘要】：目的:目前,大肠癌(Colorectal Cancer,CRC)是常见的恶性肿瘤性疾病之一,但其具体病因及发病机制仍不明确。有关报道指出多种因素如遗传背景、环境和饮食等影响了大肠癌的发病过程,其中肠道菌群结构的改变被认为与大肠癌发病风险的增加有着密切联系。有研究指出在结直肠肿瘤组织中存在大量梭杆菌属的DNA,并经过肿瘤/正常结肠组织DNA测序后得到证实。其中具核梭杆菌(Fusobacterium nucleatum,Fn)明显增加,并且与淋巴结转移呈正相关。本研究将通过C57小鼠在体构建原位大肠癌的实验动物模型,探讨Fn影响肠道肿瘤的发生及对肠道菌群结构的影响。方法:(1)Fn黏附和侵入结肠癌上皮细胞的测定。将Fn和结肠癌细胞系HCT116细胞共培养。利用HE染色进行细菌黏附、侵入细胞。基因芯片表达筛选差异基因。(2)原位大肠癌肿瘤动物模型的建立。将8-10周龄C57小鼠随机分组,经1周的环境适应期后,根据组别不同分别给予腹腔注射氧化偶氮甲烷(azoxymethane,AOM)或生理盐水,注射后7天,给予Fn菌液或生理盐水灌胃,一周后再给予第二个循环。于第81天处死小鼠,将全结肠标本取材后剖开记录肿瘤的形成情况。利用HE染色进行组织学观察,鉴别肿瘤病理性质;酶联免疫吸附试验(ELISA)检测白介素6(IL-6)、白介素8(IL-8)、环氧化酶2(COX-2)、肿瘤坏死因子(TNF-α)等,分析Fn对炎症因子的影响,从分子水平探求相关作用机制;(3)大肠癌动物模型肠道中菌群结构改变的检测。利用QIAamp DNA Stool试剂盒对肠腔粪便标本进行基因组DNA抽提;利用1%琼脂糖凝胶电泳检测抽提的基因组DNA,对各标本基因组DNA含量进行鉴定;利用ABI Gene Amp对抽提的基因组DNA进行PCR扩增,并用2%琼脂糖凝胶电泳检测PCR产物、继而荧光定量;利用Roche GS FLX 454对PCR扩增产物的16S r RNA V3可变区进行焦磷酸测序,通过SILVA数据库将所测序列进行生物信息学比对,并经Good’s覆盖率、Shannon指数、PCA、LEf Se判别分析等多种途径对各组微生物种类进行分类学和多样性的比较,以期发现肠腔菌群结构的差异性改变;(4)Fn干预下大肠癌动物模型肿瘤组织中菌群结构改变的检测及与肿瘤发生的相关实验研究。实验结束后将收集的肿瘤组织标本利用QIAamp DNA Mini试剂盒进行基因组DNA抽提,后续基因组DNA鉴定、PCR扩增、荧光定量、16S r RNA V3可变区测序和生物信息学分析均如上所述;分组比较Fn干预与对照组组织中菌群结构的差异及其与肿瘤发生情况的关系;结果:(1)Fn能黏附和侵入结肠上皮细胞,在短时间内能保持一定的黏附附和侵入量,在细胞内保持一定的活力。基因芯片筛选出差异基因,其中BIRC3和NF-k B表达明显升高。(2)实验成功建立了原位大肠癌的肿瘤动物模型。发现Fn干预下的肿瘤发生情况明显高于对照组;发现Fn组小鼠血清中的IL-6、IL-8、COX-2、TNF-α等细胞因子水平明显高于SD组,提示炎症介导了Fn促进大肠癌进展的过程;Fn组肿瘤组织PCNA、BIRC3和NF-k B水平明显高于对照组(3)Fn干预后的焦磷酸测序结果显示Fn能够导致肠道菌群结构失衡,增加大肠癌的发病风险。主成份分析(PCA)发现不同组的肠道中菌群结构存在显著差异。发现肿瘤组中硬壁菌门(Firmicutes)丰度显著增加,而拟杆菌门(Bacteroidetes)丰度明显下降;对照组中未检测到梭状杆菌门(Fusobacteria),蛋白菌门(Proteobacteria)在对照组中丰度较高,但两组间无显著性差异;在属水平上,发现肿瘤组中产丁酸盐菌Roseburia属、Eubacerium属和益生菌Ruminococcus属明显减少,而潜在致病菌Fusobacterium属则显著增加。肿瘤组中拟杆菌属(Bacteroides)丰度高于对照组(2.12%vs.0.31%,p0.001),而对照组中Lactobacillus属(60.50%vs.45.88%,p0.001)的丰度显著高于肿瘤组。结论:细胞实验证实Fn能黏附和侵入结肠上皮细胞,促进细胞增殖、抑制细胞凋亡、介导炎症过程促进肿瘤形成;通过大肠癌肿瘤动物模型发现肠道菌群结构发生了显著变化;Fn可以改变菌群结构,增加致病菌丰度,提示Fn在大肠癌发生发展中起重要的作用。另外,益生菌的应用可以降低大肠癌的发病风险。
[Abstract]:Objective: Colorectal Cancer (CRC) is one of the most common malignant diseases, but the etiology and pathogenesis of CRC are still unclear. It is reported that many factors, such as genetic background, environment and diet, affect the pathogenesis of colorectal cancer. A large number of Fusobacterium nucleatum (Fn) was found in colorectal cancer tissues and was confirmed by tumor/normal colon DNA sequencing. Fusobacterium nucleatum (Fn) was significantly increased and was positively correlated with lymph node metastasis. In this study, C57 mice were used to construct protoplasts in vivo. Methods: (1) Fn adhesion and invasion of colon cancer epithelial cells. Fn and HCT116 cells were co-cultured. HE staining was used for bacterial adhesion and invasion. Differential genes were screened by gene chip expression. C57 mice aged 8-10 weeks were randomly divided into two groups. After one week of environmental adaptation, the mice were given intraperitoneal injection of azoxymethane (AOM) or saline respectively according to the different groups. The mice were given Fn bacterial solution or saline 7 days after injection. The mice were given a second cycle one week later. Histological observation was performed by HE staining to differentiate the pathological characteristics of the tumor; enzyme-linked immunosorbent assay (ELISA) was used to detect interleukin-6 (IL-6), interleukin-8 (IL-8), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF-alpha) and so on. The effects of Fn on inflammatory factors were analyzed at molecular level. To explore the mechanism of action; (3) Detection of microbial structure in intestinal tract of animal model of colorectal cancer. Genomic DNA was extracted from intestinal faeces by QIAamp DNA Stool kit, genomic DNA was detected by 1% agarose gel electrophoresis, and the genomic DNA content of each sample was identified; the extracted gene was identified by ABI Gene Amp. Group DNA was amplified by PCR, and PCR products were detected by 2% agarose gel electrophoresis, and then quantified by fluorescence. The 16S R RNA V3 variable region of PCR products was sequenced by Roche GS FLX 454. The sequences were bioinformatically compared by SILVA database and analyzed by Good's coverage, Shannon index, PCA and LEf Se. The taxonomy and diversity of various groups of microorganisms were compared in order to discover the difference of intestinal flora structure; (4) Detection of microbial flora structure changes in animal models of colorectal cancer under Fn intervention and related experimental studies on tumorigenesis. Genomic DNA extraction, subsequent genomic DNA identification, PCR amplification, fluorescence quantitative analysis, 16S R RNA V3 variable region sequencing and bioinformatics analysis were performed with the A Mini kit. Cells, which can maintain a certain amount of adhesion and invasion in a short period of time, maintain a certain amount of activity in the cells. Gene chip screened differential genes, including BIRC3 and NF-k B expression significantly increased. (2) The experiment successfully established an animal model of colorectal cancer in situ. The levels of IL-6, IL-8, COX-2, TNF-a in serum of mice were significantly higher than those of SD group, suggesting that inflammation mediated the process of Fn promoting the progression of colorectal cancer; the levels of PCNA, BIRC 3 and NF-k B in tumor tissues of Fn group were significantly higher than those of control group (3) Pyrophosphate sequencing results showed that Fn could lead to the imbalance of intestinal flora structure and increase colorectal cancer. Principal Component Analysis (PCA) revealed significant differences in the intestinal flora structure among the different groups. Firmicutes abundance increased significantly in the tumor group, while Bacteroidetes abundance decreased significantly; Fusobacteria and Proteobacteria were not detected in the control group. The abundance of butyrate-producing bacteria Roseburia, Eubacerium and probiotics Ruminococcus decreased significantly, while the potential pathogenic bacteria Fusobacterium increased significantly. The abundance of Bacteroides in tumor group was higher than that in control group (2.12% vs. 0.31%, p0.001). The abundance of Lactobacillus (60.50% vs. 45.88%, p0.001) in group A was significantly higher than that in group B. Conclusion: Fn can adhere to and invade colon epithelial cells, promote cell proliferation, inhibit cell apoptosis, mediate inflammatory process and promote tumor formation. It can change the flora structure and increase the abundance of pathogenic bacteria, suggesting that Fn plays an important role in the occurrence and development of colorectal cancer.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.34
本文编号：2183442
[Abstract]:Objective: Colorectal Cancer (CRC) is one of the most common malignant diseases, but the etiology and pathogenesis of CRC are still unclear. It is reported that many factors, such as genetic background, environment and diet, affect the pathogenesis of colorectal cancer. A large number of Fusobacterium nucleatum (Fn) was found in colorectal cancer tissues and was confirmed by tumor/normal colon DNA sequencing. Fusobacterium nucleatum (Fn) was significantly increased and was positively correlated with lymph node metastasis. In this study, C57 mice were used to construct protoplasts in vivo. Methods: (1) Fn adhesion and invasion of colon cancer epithelial cells. Fn and HCT116 cells were co-cultured. HE staining was used for bacterial adhesion and invasion. Differential genes were screened by gene chip expression. C57 mice aged 8-10 weeks were randomly divided into two groups. After one week of environmental adaptation, the mice were given intraperitoneal injection of azoxymethane (AOM) or saline respectively according to the different groups. The mice were given Fn bacterial solution or saline 7 days after injection. The mice were given a second cycle one week later. Histological observation was performed by HE staining to differentiate the pathological characteristics of the tumor; enzyme-linked immunosorbent assay (ELISA) was used to detect interleukin-6 (IL-6), interleukin-8 (IL-8), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF-alpha) and so on. The effects of Fn on inflammatory factors were analyzed at molecular level. To explore the mechanism of action; (3) Detection of microbial structure in intestinal tract of animal model of colorectal cancer. Genomic DNA was extracted from intestinal faeces by QIAamp DNA Stool kit, genomic DNA was detected by 1% agarose gel electrophoresis, and the genomic DNA content of each sample was identified; the extracted gene was identified by ABI Gene Amp. Group DNA was amplified by PCR, and PCR products were detected by 2% agarose gel electrophoresis, and then quantified by fluorescence. The 16S R RNA V3 variable region of PCR products was sequenced by Roche GS FLX 454. The sequences were bioinformatically compared by SILVA database and analyzed by Good's coverage, Shannon index, PCA and LEf Se. The taxonomy and diversity of various groups of microorganisms were compared in order to discover the difference of intestinal flora structure; (4) Detection of microbial flora structure changes in animal models of colorectal cancer under Fn intervention and related experimental studies on tumorigenesis. Genomic DNA extraction, subsequent genomic DNA identification, PCR amplification, fluorescence quantitative analysis, 16S R RNA V3 variable region sequencing and bioinformatics analysis were performed with the A Mini kit. Cells, which can maintain a certain amount of adhesion and invasion in a short period of time, maintain a certain amount of activity in the cells. Gene chip screened differential genes, including BIRC3 and NF-k B expression significantly increased. (2) The experiment successfully established an animal model of colorectal cancer in situ. The levels of IL-6, IL-8, COX-2, TNF-a in serum of mice were significantly higher than those of SD group, suggesting that inflammation mediated the process of Fn promoting the progression of colorectal cancer; the levels of PCNA, BIRC 3 and NF-k B in tumor tissues of Fn group were significantly higher than those of control group (3) Pyrophosphate sequencing results showed that Fn could lead to the imbalance of intestinal flora structure and increase colorectal cancer. Principal Component Analysis (PCA) revealed significant differences in the intestinal flora structure among the different groups. Firmicutes abundance increased significantly in the tumor group, while Bacteroidetes abundance decreased significantly; Fusobacteria and Proteobacteria were not detected in the control group. The abundance of butyrate-producing bacteria Roseburia, Eubacerium and probiotics Ruminococcus decreased significantly, while the potential pathogenic bacteria Fusobacterium increased significantly. The abundance of Bacteroides in tumor group was higher than that in control group (2.12% vs. 0.31%, p0.001). The abundance of Lactobacillus (60.50% vs. 45.88%, p0.001) in group A was significantly higher than that in group B. Conclusion: Fn can adhere to and invade colon epithelial cells, promote cell proliferation, inhibit cell apoptosis, mediate inflammatory process and promote tumor formation. It can change the flora structure and increase the abundance of pathogenic bacteria, suggesting that Fn plays an important role in the occurrence and development of colorectal cancer.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.34
【相似文献】
相关期刊论文 前10条
1 郭杨;张玉杰;肖水清;;具核梭杆菌生物学特性及检测手段的研究进展[J];国际口腔医学杂志;2012年06期
2 倪龙兴;具核梭杆菌的生物学活性[J];牙体牙髓牙周病学杂志;1992年03期
3 黄莉莉;马瑞;夏文薇;朱彩莲;叶冬霞;;复方中药制剂对具核梭杆菌抗菌作用的体外研究[J];牙体牙髓牙周病学杂志;2010年08期
4 倪龙兴，李洁;具核梭杆菌[J];口腔医学;1994年03期
5 肖晗;葛李晨;王海芳;杜军;张革;;具核梭杆菌促进食管癌细胞增殖的研究[J];解剖学研究;2014年02期
6 薛红蕾;杨德琴;;具核梭杆菌在牙菌斑生物膜中的作用[J];国际口腔医学杂志;2013年05期
7 丁一;费晓露;徐屹;蒋俊强;肖晓蓉;;具核梭杆菌黏附和侵入上皮细胞的初步研究[J];牙体牙髓牙周病学杂志;2007年04期
8 费晓露;徐屹;丁一;肖晓蓉;蔡炜;;不同孵育时间具核梭杆菌黏附和侵入上皮细胞的变化[J];牙体牙髓牙周病学杂志;2007年07期
9 谭丽思;王宏岩;赵海礁;潘亚萍;康健;;呼吸道内具核梭杆菌与阻塞性肺疾病相关性研究[J];中国实用口腔科杂志;2013年11期
10 黄磊;徐国宾;Minghsun Liu;;自血和肝脓肿引流液培养出具核梭杆菌1例[J];临床检验杂志;2010年04期
相关会议论文 前1条
1 马瑞;夏文薇;黄莉莉;;复方中药对具核梭杆菌的抑菌作用研究[A];全国第三次牙体牙髓病学临床技术研讨会论文汇编[C];2009年
相关博士学位论文 前2条
1 李鑓;具核梭杆菌对牙龈卟啉单胞菌和伴放线聚集杆菌致病作用的影响[D];山东大学;2015年
2 郭伯敏;具核梭杆菌促进大肠癌发生的相关性研究[D];上海交通大学;2015年
相关硕士学位论文 前2条
1 王昆;具核梭杆菌诱导的肠上皮细胞炎症反应的初步研究[D];第三军医大学;2015年
2 郭杨;正畸治疗中具核梭杆菌及其毒力因子FadA粘附素的研究[D];滨州医学院;2013年
，本文编号：2183442
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2183442.html
