合并幽门螺杆菌感染的非萎缩性胃炎、萎缩伴肠上皮化生和胃癌患者的胃内菌群
发布时间：2018-10-08 06:55
【摘要】：研究背景在1983年幽门螺杆菌(H.pylori)被发现之前,胃内环境一直被认为是无菌的。经过几十年的研究,研究者发现胃内还有许多除幽门螺杆菌(H.pylori)之外的细菌存在。近年来,随着检测技术的不断进步,特别是高通量测序技术的出现,人们对胃内菌群的认识有了重大突破。胃内相对正常的菌群结构、幽门螺杆菌(H.pylori)感染对胃内菌群的影响等都被陆续报道。胃癌被认为是一种由幽门螺杆菌(H.pylori)感染、环境、遗传等多因素参与的疾病。1992年Correa医生提出了肠型胃癌的Correa级联反应模式,即肠型胃癌的发生发展经历非萎缩性胃炎、萎缩、肠上皮化生、异型增生,最终发展为胃癌,而这一模式多由幽门螺杆菌(H.pylori)感染引起。目前有证据表明,随着Correa模式的进展,幽门螺杆菌(H.pylori)在致癌方面发挥的作用逐渐减弱,提出幽门螺杆菌(H.pylori)可能只是Correa模式的诱发因素。胃酸的存在致使很多细菌都无法在胃内生存,而萎缩性胃炎和肠上皮化生患者的胃酸分泌减少,导致胃内pH值升高,进而极有可能造成某些细菌在胃内过度生长、菌群结构发生改变。目前有研究表明除幽门螺杆菌(H.pylori)外,胃内其他细菌可能也参与了胃癌的发生。但该方面的相关研究较少,且结果不一。研究目的本研究旨在分析胃内低酸的环境是否会造成胃内菌群结构发生改变,以及除幽门螺杆菌(H.pylori)之外的细菌与胃癌之间是否存在潜在关联。研究方法本研究共纳入患者22人(40个样本),其中包括合并幽门螺杆菌(H.pylori)感染的非萎缩胃炎患者10人(胃窦粘膜10块、胃体粘膜9块)、萎缩伴肠上皮化生患者6人(胃窦粘膜4块、胃体粘膜6块)和胃癌患者7人(癌组织粘膜6块,癌旁相对正常组织5块)。通过胃镜下使用无菌活检钳对患者胃粘膜进行取样获得粘膜样本。采用快速尿素酶试验、组织病理学染色及16SrDNA测序判定患者是否存在幽门螺杆菌(H.pylori)感染。使用基于IlluminaMiSeq平台的16SrDNA测序方法对胃黏膜菌群的组成结构和物种多样性展开分析。研究结果三组患者的胃粘膜菌群主要由变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、拟杆菌门(Bacteroiets)、放线菌门(Actinobacteria)、梭杆菌门(Fusobacteria)和一种无法分类到菌门的细菌(unclassified-k-norank-d-Bacteria)组成,其中变形菌门(Proteobacteria占 70%以上。进行组间物种差异性分析发现:幽门螺杆菌(H.pylori)在萎缩伴肠上皮化生患者的胃内菌群中所占比例最高,胃癌患者中最低;无论是将胃癌组织还是癌旁相对正常组织的菌群与非萎缩性胃炎患者及萎缩伴肠上皮化生患者的胃粘膜菌群做对比,都只有该未分类到门的细菌(unclassified-k-norank-d-Bacteria)的改变具有统计学意义,该细菌从非萎缩性胃炎到萎缩伴肠上皮化生再到胃癌组呈递减趋势;除幽门螺杆菌(H.pylori)外,其他细菌在萎缩伴肠上皮化生患者胃粘膜的定植均少于非萎缩性胃炎患者;未见有细菌随Correa序列进展呈递增趋势;胃癌组织与癌旁相对正常组织的菌群结构尽管有所不同,但并发现物种间的差异具有统计学意义。α多样性方面:无论是将胃癌组织还是癌旁相对正常组织的菌群与另外两组做对比,从非萎缩性胃炎到萎缩伴肠上皮化生再到胃癌组,胃内菌群的丰富度都逐渐降低,萎缩伴肠上皮化生患者胃粘膜物种的多样性都是最低的;虽胃癌组织上菌群的物种多样性高于非萎缩性胃炎组,但癌旁相对正常组织的物种多样性则与非萎缩性胃炎组几乎没有差异。胃癌患者癌组织菌群的多样性和丰富度都高于癌旁相对正常组织。β多样性方面:仅有非萎缩性胃炎组和胃癌组在NMDS1方向呈明显分离趋势,而萎缩伴肠上皮化生组散在分布于两组之间。研究结论对比合并幽门螺杆菌(H.pyliri)感染的非萎缩性胃炎及胃癌患者,萎缩伴肠上皮化生患者胃内细菌并未呈过度生长趋势,幽门螺杆菌(H.pylori)可能仍是被其感染的患者中导致胃癌的最重要的因素。胃癌组织上可能存在一些继发性生长的细菌。下一步研究不合并幽门螺杆菌(H.pylori)感染的处于Correa序列中不同阶段的患者、上皮内瘤变或早期胃癌患者的胃内菌群亦有重要意义。此外,宏基因组测序对于进行胃内菌群的功能验证十分重要,有待于在将来的研究中开展。研究意义本研究选取了合并幽门螺杆菌(H.pylori)感染的Correa序列中最具有代表性的非萎缩性胃炎、萎缩伴肠上皮化生和胃癌患者的胃粘膜样本进行测序分析,为国内首次。本研究同时采用传统的检测方法(快速尿素酶试验和组织病理学染色)和测序法判定有无幽门螺杆菌(H.pylori)感染,减少假阴性,提高了准确率。本研究对于揭示合并幽门螺杆菌(H.pylori)感染的患者在从非萎缩性胃炎到发展至胃癌的过程中幽门螺杆菌(H.pylori)及其他胃内菌群所发挥的作用有重要意义。
[Abstract]:The context of the study was considered sterile before Helicobacter pylori (H. pylori) was found in 1983. After decades of research, researchers found that there are many bacteria other than Helicobacter pylori (H.pylori) in the stomach. In recent years, with the progress of detection technology, especially the emergence of high-throughput sequencing technology, there has been a major breakthrough in understanding the flora of the stomach. The relative normal flora structure of the stomach, the influence of H. pylori infection on the intragastric flora and so on are reported in succession. Gastric cancer is considered to be a disease caused by multiple factors such as Helicobacter pylori (H. pylori) infection, environment, heredity and so on. In 1992, Dr. Correa proposed the Correa cascade reaction model of intestinal type stomach cancer, namely, the development of intestinal type gastric cancer experienced non-atrophic gastritis, atrophy, intestinal metaplasia and heterotypic hyperplasia. Final development is gastric cancer, and this pattern is caused by Helicobacter pylori (H. pylori) infection. There is currently evidence that Helicobacter pylori (H. pylori) may only be a contributing factor in Correa mode with the progression of Correa's model and the diminishing role of H. pylori in carcinogenesis. The presence of gastric acid causes many bacteria to survive in the stomach, while the gastric acid secretion in atrophic gastritis and intestinal metaplasia is reduced, resulting in a rise in pH in the stomach, which in turn may cause some bacteria to grow in the stomach and the structure of the flora changes. Studies have shown that in addition to Helicobacter pylori (H.pylori), other bacteria in the stomach may also be involved in the occurrence of gastric cancer. However, there are few related studies in this area, and the results are not one. The purpose of this study was to analyze the presence or absence of a potential association between bacteria in the stomach and gastric cancer, in addition to Helicobacter pylori (H.pylori). A total of 22 patients (40 samples) were enrolled in this study, including 10 patients with non-atrophic gastritis infected with Helicobacter pylori (H. pylori) (10 blocks of gastric mucosa, 9 gastric mucosa), 6 patients with atrophy accompanied by intestinal metaplasia (4 blocks of intestinal mucosa), Gastric body mucosa (6 blocks) and gastric cancer patients (6 blocks of cancer tissue mucosa, 5 normal tissues adjacent to cancer). The mucosa samples were obtained by sampling the gastric mucosa of the patient using a sterile biopsy forceps under gastroscope. It was determined whether Helicobacter pylori (H. pylori) infection was present in patients by rapid urea enzyme test, histopathological staining and 16SrDNA sequencing. The composition structure and species diversity of gastric mucosa flora were analyzed using the 16SrDNA sequencing method based on the Illumina MiSeq platform. Results Three groups of gastric mucosal flora were mainly composed of Proteobacter, Firmicutes, Bacteroiets, Actinobacillus, Clostridium and a bacterium which could not be classified into bacteria gate, among which Proteobacteria accounted for more than 70%. It was found that Helicobacter pylori (H. pylori) was the highest among the patients with intestinal metaplasia with intestinal metaplasia and the lowest in gastric cancer patients. whether gastric cancer tissue or a group of non-atrophic gastritis and a gastric mucosal flora of a non-atrophic gastritis patient and a metaplasia of the intestinal metaplasia have a statistical significance only when compared with the gastric mucosal flora of patients with non-atrophic gastritis and metaplasia of intestinal metaplasia, In addition to Helicobacter pylori (H. pylori), the colonization of gastric mucosa was less than that of non-atrophic gastritis. There was no increase in the progression of the bacteria with the Correa sequence; however, the structure of the bacteria group of the gastric cancer tissues and the normal tissues adjacent to the cancer was different, but it was found that the differences among the species were statistically significant. In terms of the diversity of gastric cancer, whether gastric cancer tissue or normal tissue beside cancer was compared with the other two groups, the richness of intragastric flora decreased gradually from non-atrophic gastritis to atrophy and intestinal metaplasia to gastric cancer group. The diversity of gastric mucosal species in patients with atrophy and intestinal metaplasia was the lowest. Although the species diversity of bacteria in gastric cancer tissues was higher than that of non-atrophic gastritis, there was little difference between the species diversity of normal tissues and the non-atrophic gastritis group. The diversity and richness of tissue flora in patients with gastric cancer were higher than that of normal tissues adjacent to cancer. In terms of diversity, there were only non-atrophic gastritis group and gastric cancer group in NMDS1 direction, while atrophy accompanied with intestinal metaplasia was scattered between the two groups. The study concluded that Helicobacter pylori (H. pylori) may still be the most important factor leading to gastric cancer in patients with non-atrophic gastritis and gastric cancer infected by H. pyliri. There may be some secondary growing bacteria on gastric cancer tissue. The next study was also important to study the intragastric flora of patients with different stages of the Correa sequence infected with Helicobacter pylori (H. pylori), or in patients with intraepithelial neoplasia or early gastric cancer. In addition, macro-genome sequencing is important for the functional verification of intragastric flora, which is to be done in future studies. In this study, the most representative non-atrophic gastritis, atrophy accompanying intestinal metaplasia and gastric mucosal samples of gastric cancer patients with Helicobacter pylori (H. pylori) infection were selected for the first time in China. At the same time, traditional detection methods (rapid urea enzyme test and histopathology staining) and sequencing were used to determine the presence or absence of Helicobacter pylori (H. pylori) infection, to reduce false negative and to improve the accuracy. This study is of great significance to reveal the role played by H. pylori and other intragastric flora in patients infected with Helicobacter pylori (H.pylori) from non-atrophic gastritis to gastric cancer.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R573;R735.2
本文编号：2255851
[Abstract]:The context of the study was considered sterile before Helicobacter pylori (H. pylori) was found in 1983. After decades of research, researchers found that there are many bacteria other than Helicobacter pylori (H.pylori) in the stomach. In recent years, with the progress of detection technology, especially the emergence of high-throughput sequencing technology, there has been a major breakthrough in understanding the flora of the stomach. The relative normal flora structure of the stomach, the influence of H. pylori infection on the intragastric flora and so on are reported in succession. Gastric cancer is considered to be a disease caused by multiple factors such as Helicobacter pylori (H. pylori) infection, environment, heredity and so on. In 1992, Dr. Correa proposed the Correa cascade reaction model of intestinal type stomach cancer, namely, the development of intestinal type gastric cancer experienced non-atrophic gastritis, atrophy, intestinal metaplasia and heterotypic hyperplasia. Final development is gastric cancer, and this pattern is caused by Helicobacter pylori (H. pylori) infection. There is currently evidence that Helicobacter pylori (H. pylori) may only be a contributing factor in Correa mode with the progression of Correa's model and the diminishing role of H. pylori in carcinogenesis. The presence of gastric acid causes many bacteria to survive in the stomach, while the gastric acid secretion in atrophic gastritis and intestinal metaplasia is reduced, resulting in a rise in pH in the stomach, which in turn may cause some bacteria to grow in the stomach and the structure of the flora changes. Studies have shown that in addition to Helicobacter pylori (H.pylori), other bacteria in the stomach may also be involved in the occurrence of gastric cancer. However, there are few related studies in this area, and the results are not one. The purpose of this study was to analyze the presence or absence of a potential association between bacteria in the stomach and gastric cancer, in addition to Helicobacter pylori (H.pylori). A total of 22 patients (40 samples) were enrolled in this study, including 10 patients with non-atrophic gastritis infected with Helicobacter pylori (H. pylori) (10 blocks of gastric mucosa, 9 gastric mucosa), 6 patients with atrophy accompanied by intestinal metaplasia (4 blocks of intestinal mucosa), Gastric body mucosa (6 blocks) and gastric cancer patients (6 blocks of cancer tissue mucosa, 5 normal tissues adjacent to cancer). The mucosa samples were obtained by sampling the gastric mucosa of the patient using a sterile biopsy forceps under gastroscope. It was determined whether Helicobacter pylori (H. pylori) infection was present in patients by rapid urea enzyme test, histopathological staining and 16SrDNA sequencing. The composition structure and species diversity of gastric mucosa flora were analyzed using the 16SrDNA sequencing method based on the Illumina MiSeq platform. Results Three groups of gastric mucosal flora were mainly composed of Proteobacter, Firmicutes, Bacteroiets, Actinobacillus, Clostridium and a bacterium which could not be classified into bacteria gate, among which Proteobacteria accounted for more than 70%. It was found that Helicobacter pylori (H. pylori) was the highest among the patients with intestinal metaplasia with intestinal metaplasia and the lowest in gastric cancer patients. whether gastric cancer tissue or a group of non-atrophic gastritis and a gastric mucosal flora of a non-atrophic gastritis patient and a metaplasia of the intestinal metaplasia have a statistical significance only when compared with the gastric mucosal flora of patients with non-atrophic gastritis and metaplasia of intestinal metaplasia, In addition to Helicobacter pylori (H. pylori), the colonization of gastric mucosa was less than that of non-atrophic gastritis. There was no increase in the progression of the bacteria with the Correa sequence; however, the structure of the bacteria group of the gastric cancer tissues and the normal tissues adjacent to the cancer was different, but it was found that the differences among the species were statistically significant. In terms of the diversity of gastric cancer, whether gastric cancer tissue or normal tissue beside cancer was compared with the other two groups, the richness of intragastric flora decreased gradually from non-atrophic gastritis to atrophy and intestinal metaplasia to gastric cancer group. The diversity of gastric mucosal species in patients with atrophy and intestinal metaplasia was the lowest. Although the species diversity of bacteria in gastric cancer tissues was higher than that of non-atrophic gastritis, there was little difference between the species diversity of normal tissues and the non-atrophic gastritis group. The diversity and richness of tissue flora in patients with gastric cancer were higher than that of normal tissues adjacent to cancer. In terms of diversity, there were only non-atrophic gastritis group and gastric cancer group in NMDS1 direction, while atrophy accompanied with intestinal metaplasia was scattered between the two groups. The study concluded that Helicobacter pylori (H. pylori) may still be the most important factor leading to gastric cancer in patients with non-atrophic gastritis and gastric cancer infected by H. pyliri. There may be some secondary growing bacteria on gastric cancer tissue. The next study was also important to study the intragastric flora of patients with different stages of the Correa sequence infected with Helicobacter pylori (H. pylori), or in patients with intraepithelial neoplasia or early gastric cancer. In addition, macro-genome sequencing is important for the functional verification of intragastric flora, which is to be done in future studies. In this study, the most representative non-atrophic gastritis, atrophy accompanying intestinal metaplasia and gastric mucosal samples of gastric cancer patients with Helicobacter pylori (H. pylori) infection were selected for the first time in China. At the same time, traditional detection methods (rapid urea enzyme test and histopathology staining) and sequencing were used to determine the presence or absence of Helicobacter pylori (H. pylori) infection, to reduce false negative and to improve the accuracy. This study is of great significance to reveal the role played by H. pylori and other intragastric flora in patients infected with Helicobacter pylori (H.pylori) from non-atrophic gastritis to gastric cancer.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R573;R735.2
【参考文献】
相关期刊论文 前2条
1 施宏;谢招飞;黄贺;黄瑞;蔡和利;林楷;郑卫;;内镜诊断胃幽门螺杆菌感染的临床研究[J];中华消化杂志;2016年11期
2 ;Classification of histological severity of Helicobacter pylori-associated gastritis by confocal laser endomicroscopy[J];World Journal of Gastroenterology;2010年41期
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