具核梭桿菌促進(jìn)大腸癌發(fā)生的相關(guān)性研究
發(fā)布時(shí)間：2018-08-14 16:27
【摘要】：目的:目前,大腸癌(Colorectal Cancer,CRC)是常見的惡性腫瘤性疾病之一,但其具體病因及發(fā)病機(jī)制仍不明確。有關(guān)報(bào)道指出多種因素如遺傳背景、環(huán)境和飲食等影響了大腸癌的發(fā)病過(guò)程,其中腸道菌群結(jié)構(gòu)的改變被認(rèn)為與大腸癌發(fā)病風(fēng)險(xiǎn)的增加有著密切聯(lián)系。有研究指出在結(jié)直腸腫瘤組織中存在大量梭桿菌屬的DNA,并經(jīng)過(guò)腫瘤/正常結(jié)腸組織DNA測(cè)序后得到證實(shí)。其中具核梭桿菌(Fusobacterium nucleatum,Fn)明顯增加,并且與淋巴結(jié)轉(zhuǎn)移呈正相關(guān)。本研究將通過(guò)C57小鼠在體構(gòu)建原位大腸癌的實(shí)驗(yàn)動(dòng)物模型,探討Fn影響腸道腫瘤的發(fā)生及對(duì)腸道菌群結(jié)構(gòu)的影響。方法:(1)Fn黏附和侵入結(jié)腸癌上皮細(xì)胞的測(cè)定。將Fn和結(jié)腸癌細(xì)胞系HCT116細(xì)胞共培養(yǎng)。利用HE染色進(jìn)行細(xì)菌黏附、侵入細(xì)胞。基因芯片表達(dá)篩選差異基因。(2)原位大腸癌腫瘤動(dòng)物模型的建立。將8-10周齡C57小鼠隨機(jī)分組,經(jīng)1周的環(huán)境適應(yīng)期后,根據(jù)組別不同分別給予腹腔注射氧化偶氮甲烷(azoxymethane,AOM)或生理鹽水,注射后7天,給予Fn菌液或生理鹽水灌胃,一周后再給予第二個(gè)循環(huán)。于第81天處死小鼠,將全結(jié)腸標(biāo)本取材后剖開記錄腫瘤的形成情況。利用HE染色進(jìn)行組織學(xué)觀察,鑒別腫瘤病理性質(zhì);酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測(cè)白介素6(IL-6)、白介素8(IL-8)、環(huán)氧化酶2(COX-2)、腫瘤壞死因子(TNF-α)等,分析Fn對(duì)炎癥因子的影響,從分子水平探求相關(guān)作用機(jī)制;(3)大腸癌動(dòng)物模型腸道中菌群結(jié)構(gòu)改變的檢測(cè)。利用QIAamp DNA Stool試劑盒對(duì)腸腔糞便標(biāo)本進(jìn)行基因組DNA抽提;利用1%瓊脂糖凝膠電泳檢測(cè)抽提的基因組DNA,對(duì)各標(biāo)本基因組DNA含量進(jìn)行鑒定;利用ABI Gene Amp對(duì)抽提的基因組DNA進(jìn)行PCR擴(kuò)增,并用2%瓊脂糖凝膠電泳檢測(cè)PCR產(chǎn)物、繼而熒光定量;利用Roche GS FLX 454對(duì)PCR擴(kuò)增產(chǎn)物的16S r RNA V3可變區(qū)進(jìn)行焦磷酸測(cè)序,通過(guò)SILVA數(shù)據(jù)庫(kù)將所測(cè)序列進(jìn)行生物信息學(xué)比對(duì),并經(jīng)Good’s覆蓋率、Shannon指數(shù)、PCA、LEf Se判別分析等多種途徑對(duì)各組微生物種類進(jìn)行分類學(xué)和多樣性的比較,以期發(fā)現(xiàn)腸腔菌群結(jié)構(gòu)的差異性改變;(4)Fn干預(yù)下大腸癌動(dòng)物模型腫瘤組織中菌群結(jié)構(gòu)改變的檢測(cè)及與腫瘤發(fā)生的相關(guān)實(shí)驗(yàn)研究。實(shí)驗(yàn)結(jié)束后將收集的腫瘤組織標(biāo)本利用QIAamp DNA Mini試劑盒進(jìn)行基因組DNA抽提,后續(xù)基因組DNA鑒定、PCR擴(kuò)增、熒光定量、16S r RNA V3可變區(qū)測(cè)序和生物信息學(xué)分析均如上所述;分組比較Fn干預(yù)與對(duì)照組組織中菌群結(jié)構(gòu)的差異及其與腫瘤發(fā)生情況的關(guān)系;結(jié)果:(1)Fn能黏附和侵入結(jié)腸上皮細(xì)胞,在短時(shí)間內(nèi)能保持一定的黏附附和侵入量,在細(xì)胞內(nèi)保持一定的活力。基因芯片篩選出差異基因,其中BIRC3和NF-k B表達(dá)明顯升高。(2)實(shí)驗(yàn)成功建立了原位大腸癌的腫瘤動(dòng)物模型。發(fā)現(xiàn)Fn干預(yù)下的腫瘤發(fā)生情況明顯高于對(duì)照組;發(fā)現(xiàn)Fn組小鼠血清中的IL-6、IL-8、COX-2、TNF-α等細(xì)胞因子水平明顯高于SD組,提示炎癥介導(dǎo)了Fn促進(jìn)大腸癌進(jìn)展的過(guò)程;Fn組腫瘤組織PCNA、BIRC3和NF-k B水平明顯高于對(duì)照組(3)Fn干預(yù)后的焦磷酸測(cè)序結(jié)果顯示Fn能夠?qū)е履c道菌群結(jié)構(gòu)失衡,增加大腸癌的發(fā)病風(fēng)險(xiǎn)。主成份分析(PCA)發(fā)現(xiàn)不同組的腸道中菌群結(jié)構(gòu)存在顯著差異。發(fā)現(xiàn)腫瘤組中硬壁菌門(Firmicutes)豐度顯著增加,而擬桿菌門(Bacteroidetes)豐度明顯下降;對(duì)照組中未檢測(cè)到梭狀桿菌門(Fusobacteria),蛋白菌門(Proteobacteria)在對(duì)照組中豐度較高,但兩組間無(wú)顯著性差異;在屬水平上,發(fā)現(xiàn)腫瘤組中產(chǎn)丁酸鹽菌Roseburia屬、Eubacerium屬和益生菌Ruminococcus屬明顯減少,而潛在致病菌Fusobacterium屬則顯著增加。腫瘤組中擬桿菌屬(Bacteroides)豐度高于對(duì)照組(2.12%vs.0.31%,p0.001),而對(duì)照組中Lactobacillus屬(60.50%vs.45.88%,p0.001)的豐度顯著高于腫瘤組。結(jié)論:細(xì)胞實(shí)驗(yàn)證實(shí)Fn能黏附和侵入結(jié)腸上皮細(xì)胞,促進(jìn)細(xì)胞增殖、抑制細(xì)胞凋亡、介導(dǎo)炎癥過(guò)程促進(jìn)腫瘤形成;通過(guò)大腸癌腫瘤動(dòng)物模型發(fā)現(xiàn)腸道菌群結(jié)構(gòu)發(fā)生了顯著變化;Fn可以改變菌群結(jié)構(gòu),增加致病菌豐度,提示Fn在大腸癌發(fā)生發(fā)展中起重要的作用。另外,益生菌的應(yīng)用可以降低大腸癌的發(fā)病風(fēng)險(xiǎn)。
[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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.34
本文編號(hào)：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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.34
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