【摘要】：研究背景在1983年幽門(mén)螺桿菌(H.pylori)被發(fā)現(xiàn)之前,胃內(nèi)環(huán)境一直被認(rèn)為是無(wú)菌的。經(jīng)過(guò)幾十年的研究,研究者發(fā)現(xiàn)胃內(nèi)還有許多除幽門(mén)螺桿菌(H.pylori)之外的細(xì)菌存在。近年來(lái),隨著檢測(cè)技術(shù)的不斷進(jìn)步,特別是高通量測(cè)序技術(shù)的出現(xiàn),人們對(duì)胃內(nèi)菌群的認(rèn)識(shí)有了重大突破。胃內(nèi)相對(duì)正常的菌群結(jié)構(gòu)、幽門(mén)螺桿菌(H.pylori)感染對(duì)胃內(nèi)菌群的影響等都被陸續(xù)報(bào)道。胃癌被認(rèn)為是一種由幽門(mén)螺桿菌(H.pylori)感染、環(huán)境、遺傳等多因素參與的疾病。1992年Correa醫(yī)生提出了腸型胃癌的Correa級(jí)聯(lián)反應(yīng)模式,即腸型胃癌的發(fā)生發(fā)展經(jīng)歷非萎縮性胃炎、萎縮、腸上皮化生、異型增生,最終發(fā)展為胃癌,而這一模式多由幽門(mén)螺桿菌(H.pylori)感染引起。目前有證據(jù)表明,隨著Correa模式的進(jìn)展,幽門(mén)螺桿菌(H.pylori)在致癌方面發(fā)揮的作用逐漸減弱,提出幽門(mén)螺桿菌(H.pylori)可能只是Correa模式的誘發(fā)因素。胃酸的存在致使很多細(xì)菌都無(wú)法在胃內(nèi)生存,而萎縮性胃炎和腸上皮化生患者的胃酸分泌減少,導(dǎo)致胃內(nèi)pH值升高,進(jìn)而極有可能造成某些細(xì)菌在胃內(nèi)過(guò)度生長(zhǎng)、菌群結(jié)構(gòu)發(fā)生改變。目前有研究表明除幽門(mén)螺桿菌(H.pylori)外,胃內(nèi)其他細(xì)菌可能也參與了胃癌的發(fā)生。但該方面的相關(guān)研究較少,且結(jié)果不一。研究目的本研究旨在分析胃內(nèi)低酸的環(huán)境是否會(huì)造成胃內(nèi)菌群結(jié)構(gòu)發(fā)生改變,以及除幽門(mén)螺桿菌(H.pylori)之外的細(xì)菌與胃癌之間是否存在潛在關(guān)聯(lián)。研究方法本研究共納入患者22人(40個(gè)樣本),其中包括合并幽門(mén)螺桿菌(H.pylori)感染的非萎縮胃炎患者10人(胃竇粘膜10塊、胃體粘膜9塊)、萎縮伴腸上皮化生患者6人(胃竇粘膜4塊、胃體粘膜6塊)和胃癌患者7人(癌組織粘膜6塊,癌旁相對(duì)正常組織5塊)。通過(guò)胃鏡下使用無(wú)菌活檢鉗對(duì)患者胃粘膜進(jìn)行取樣獲得粘膜樣本。采用快速尿素酶試驗(yàn)、組織病理學(xué)染色及16SrDNA測(cè)序判定患者是否存在幽門(mén)螺桿菌(H.pylori)感染。使用基于IlluminaMiSeq平臺(tái)的16SrDNA測(cè)序方法對(duì)胃黏膜菌群的組成結(jié)構(gòu)和物種多樣性展開(kāi)分析。研究結(jié)果三組患者的胃粘膜菌群主要由變形菌門(mén)(Proteobacteria)、厚壁菌門(mén)(Firmicutes)、擬桿菌門(mén)(Bacteroiets)、放線菌門(mén)(Actinobacteria)、梭桿菌門(mén)(Fusobacteria)和一種無(wú)法分類(lèi)到菌門(mén)的細(xì)菌(unclassified-k-norank-d-Bacteria)組成,其中變形菌門(mén)(Proteobacteria占 70%以上。進(jìn)行組間物種差異性分析發(fā)現(xiàn):幽門(mén)螺桿菌(H.pylori)在萎縮伴腸上皮化生患者的胃內(nèi)菌群中所占比例最高,胃癌患者中最低;無(wú)論是將胃癌組織還是癌旁相對(duì)正常組織的菌群與非萎縮性胃炎患者及萎縮伴腸上皮化生患者的胃粘膜菌群做對(duì)比,都只有該未分類(lèi)到門(mén)的細(xì)菌(unclassified-k-norank-d-Bacteria)的改變具有統(tǒng)計(jì)學(xué)意義,該細(xì)菌從非萎縮性胃炎到萎縮伴腸上皮化生再到胃癌組呈遞減趨勢(shì);除幽門(mén)螺桿菌(H.pylori)外,其他細(xì)菌在萎縮伴腸上皮化生患者胃粘膜的定植均少于非萎縮性胃炎患者;未見(jiàn)有細(xì)菌隨Correa序列進(jìn)展呈遞增趨勢(shì);胃癌組織與癌旁相對(duì)正常組織的菌群結(jié)構(gòu)盡管有所不同,但并發(fā)現(xiàn)物種間的差異具有統(tǒng)計(jì)學(xué)意義。α多樣性方面:無(wú)論是將胃癌組織還是癌旁相對(duì)正常組織的菌群與另外兩組做對(duì)比,從非萎縮性胃炎到萎縮伴腸上皮化生再到胃癌組,胃內(nèi)菌群的豐富度都逐漸降低,萎縮伴腸上皮化生患者胃粘膜物種的多樣性都是最低的;雖胃癌組織上菌群的物種多樣性高于非萎縮性胃炎組,但癌旁相對(duì)正常組織的物種多樣性則與非萎縮性胃炎組幾乎沒(méi)有差異。胃癌患者癌組織菌群的多樣性和豐富度都高于癌旁相對(duì)正常組織。β多樣性方面:僅有非萎縮性胃炎組和胃癌組在NMDS1方向呈明顯分離趨勢(shì),而萎縮伴腸上皮化生組散在分布于兩組之間。研究結(jié)論對(duì)比合并幽門(mén)螺桿菌(H.pyliri)感染的非萎縮性胃炎及胃癌患者,萎縮伴腸上皮化生患者胃內(nèi)細(xì)菌并未呈過(guò)度生長(zhǎng)趨勢(shì),幽門(mén)螺桿菌(H.pylori)可能仍是被其感染的患者中導(dǎo)致胃癌的最重要的因素。胃癌組織上可能存在一些繼發(fā)性生長(zhǎng)的細(xì)菌。下一步研究不合并幽門(mén)螺桿菌(H.pylori)感染的處于Correa序列中不同階段的患者、上皮內(nèi)瘤變或早期胃癌患者的胃內(nèi)菌群亦有重要意義。此外,宏基因組測(cè)序?qū)τ谶M(jìn)行胃內(nèi)菌群的功能驗(yàn)證十分重要,有待于在將來(lái)的研究中開(kāi)展。研究意義本研究選取了合并幽門(mén)螺桿菌(H.pylori)感染的Correa序列中最具有代表性的非萎縮性胃炎、萎縮伴腸上皮化生和胃癌患者的胃粘膜樣本進(jìn)行測(cè)序分析,為國(guó)內(nèi)首次。本研究同時(shí)采用傳統(tǒng)的檢測(cè)方法(快速尿素酶試驗(yàn)和組織病理學(xué)染色)和測(cè)序法判定有無(wú)幽門(mén)螺桿菌(H.pylori)感染,減少假陰性,提高了準(zhǔn)確率。本研究對(duì)于揭示合并幽門(mén)螺桿菌(H.pylori)感染的患者在從非萎縮性胃炎到發(fā)展至胃癌的過(guò)程中幽門(mén)螺桿菌(H.pylori)及其他胃內(nèi)菌群所發(fā)揮的作用有重要意義。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R573;R735.2

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相關(guān)期刊論文 前2條

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本文編號(hào)：2255851