【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)病機制仍不明確。有關(guān)報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)病過程,其中腸道菌群結(jié)構(gòu)的改變被認為與大腸癌發(fā)病風險的增加有著密切聯(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)過腫瘤/正常結(jié)腸組織DNA測序后得到證實。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C57小鼠在體構(gòu)建原位大腸癌的實驗動物模型,探討Fn影響腸道腫瘤的發(fā)生及對腸道菌群結(jié)構(gòu)的影響。方法:(1)Fn黏附和侵入結(jié)腸癌上皮細胞的測定。將Fn和結(jié)腸癌細胞系HCT116細胞共培養(yǎng)。利用HE染色進行細菌黏附、侵入細胞。基因芯片表達篩選差異基因。(2)原位大腸癌腫瘤動物模型的建立。將8-10周齡C57小鼠隨機分組,經(jīng)1周的環(huán)境適應期后,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循環(huán)。于第81天處死小鼠,將全結(jié)腸標本取材后剖開記錄腫瘤的形成情況。利用HE染色進行組織學觀察,鑒別腫瘤病理性質(zhì);酶聯(lián)免疫吸附試驗(ELISA)檢測白介素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對炎癥因子的影響,從分子水平探求相關(guān)作用機制;(3)大腸癌動物模型腸道中菌群結(jié)構(gòu)改變的檢測。利用QIAamp DNA Stool試劑盒對腸腔糞便標本進行基因組DNA抽提;利用1%瓊脂糖凝膠電泳檢測抽提的基因組DNA,對各標本基因組DNA含量進行鑒定;利用ABI Gene Amp對抽提的基因組DNA進行PCR擴增,并用2%瓊脂糖凝膠電泳檢測PCR產(chǎn)物、繼而熒光定量;利用Roche GS FLX 454對PCR擴增產(chǎn)物的16S r RNA V3可變區(qū)進行焦磷酸測序,通過SILVA數(shù)據(jù)庫將所測序列進行生物信息學比對,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各組微生物種類進行分類學和多樣性的比較,以期發(fā)現(xiàn)腸腔菌群結(jié)構(gòu)的差異性改變;(4)Fn干預下大腸癌動物模型腫瘤組織中菌群結(jié)構(gòu)改變的檢測及與腫瘤發(fā)生的相關(guān)實驗研究。實驗結(jié)束后將收集的腫瘤組織標本利用QIAamp DNA Mini試劑盒進行基因組DNA抽提,后續(xù)基因組DNA鑒定、PCR擴增、熒光定量、16S r RNA V3可變區(qū)測序和生物信息學分析均如上所述;分組比較Fn干預與對照組組織中菌群結(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)腸上皮細胞,在短時間內(nèi)能保持一定的黏附附和侵入量,在細胞內(nèi)保持一定的活力�；蛐酒Y選出差異基因,其中BIRC3和NF-k B表達明顯升高。(2)實驗成功建立了原位大腸癌的腫瘤動物模型。發(fā)現(xiàn)Fn干預下的腫瘤發(fā)生情況明顯高于對照組;發(fā)現(xiàn)Fn組小鼠血清中的IL-6、IL-8、COX-2、TNF-α等細胞因子水平明顯高于SD組,提示炎癥介導了Fn促進大腸癌進展的過程;Fn組腫瘤組織PCNA、BIRC3和NF-k B水平明顯高于對照組(3)Fn干預后的焦磷酸測序結(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)病風險。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;對照組中未檢測到梭狀桿菌門(Fusobacteria),蛋白菌門(Proteobacteria)在對照組中豐度較高,但兩組間無顯著性差異;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)豐度高于對照組(2.12%vs.0.31%,p0.001),而對照組中Lactobacillus屬(60.50%vs.45.88%,p0.001)的豐度顯著高于腫瘤組。結(jié)論:細胞實驗證實Fn能黏附和侵入結(jié)腸上皮細胞,促進細胞增殖、抑制細胞凋亡、介導炎癥過程促進腫瘤形成;通過大腸癌腫瘤動物模型發(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,益生菌的應用可以降低大腸癌的發(fā)病風險。
[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.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.34

【相似文獻】

相關(guān)期刊論文 前10條

1 郭楊;張玉杰;肖水清;;具核梭桿菌生物學特性及檢測手段的研究進展[J];國際口腔醫(yī)學雜志;2012年06期

2 倪龍興;具核梭桿菌的生物學活性[J];牙體牙髓牙周病學雜志;1992年03期

3 黃莉莉;馬瑞;夏文薇;朱彩蓮;葉冬霞;;復方中藥制劑對具核梭桿菌抗菌作用的體外研究[J];牙體牙髓牙周病學雜志;2010年08期

4 倪龍興，李潔;具核梭桿菌[J];口腔醫(yī)學;1994年03期

5 肖晗;葛李晨;王海芳;杜軍;張革;;具核梭桿菌促進食管癌細胞增殖的研究[J];解剖學研究;2014年02期

6 薛紅蕾;楊德琴;;具核梭桿菌在牙菌斑生物膜中的作用[J];國際口腔醫(yī)學雜志;2013年05期

7 丁一;費曉露;徐屹;蔣俊強;肖曉蓉;;具核梭桿菌黏附和侵入上皮細胞的初步研究[J];牙體牙髓牙周病學雜志;2007年04期

8 費曉露;徐屹;丁一;肖曉蓉;蔡煒;;不同孵育時間具核梭桿菌黏附和侵入上皮細胞的變化[J];牙體牙髓牙周病學雜志;2007年07期

9 譚麗思;王宏巖;趙海礁;潘亞萍;康健;;呼吸道內(nèi)具核梭桿菌與阻塞性肺疾病相關(guān)性研究[J];中國實用口腔科雜志;2013年11期

10 黃磊;徐國賓;Minghsun Liu;;自血和肝膿腫引流液培養(yǎng)出具核梭桿菌1例[J];臨床檢驗雜志;2010年04期

相關(guān)會議論文 前1條

1 馬瑞;夏文薇;黃莉莉;;復方中藥對具核梭桿菌的抑菌作用研究[A];全國第三次牙體牙髓病學臨床技術(shù)研討會論文匯編[C];2009年

相關(guān)博士學位論文 前2條

1 李鑓;具核梭桿菌對牙齦卟啉單胞菌和伴放線聚集桿菌致病作用的影響[D];山東大學;2015年

2 郭伯敏;具核梭桿菌促進大腸癌發(fā)生的相關(guān)性研究[D];上海交通大學;2015年

相關(guān)碩士學位論文 前2條

1 王昆;具核梭桿菌誘導的腸上皮細胞炎癥反應的初步研究[D];第三軍醫(yī)大學;2015年

2 郭楊;正畸治療中具核梭桿菌及其毒力因子FadA粘附素的研究[D];濱州醫(yī)學院;2013年

，

本文編號：2183442