【摘要】：背景胃癌是世界范圍內(nèi)最常見的惡性腫瘤之一,其死亡率居于全球癌癥相關(guān)死亡率的第三位。中國是胃癌高發(fā)國家,中國胃癌發(fā)病率居世界第二,僅次于日本。胃癌的發(fā)生是多因素共同作用的結(jié)果,其中幽門螺桿菌(Helicobacter pylori, H. pylori)感染是胃癌的重要致病因素。雖然已有大量研究證實(shí)幽門螺桿菌感染與胃癌發(fā)生之間的關(guān)系,但其確切的致病機(jī)制仍不明確。近期研究發(fā)現(xiàn),幽門螺桿菌感染可以調(diào)控宿主細(xì)胞的表觀遺傳學(xué)修飾的改變,例如組蛋白修飾狀態(tài)的改變、DNA甲基化等,這些改變與幽門螺桿菌的致癌作用密切相關(guān)。組蛋白去甲基化酶JMJD2B,又稱KDM4B,是新近發(fā)現(xiàn)的JMJD2蛋白家族中的一員,主要靶向組蛋白H3第9位賴氨酸的三甲基(H3K9me3)使其發(fā)生去甲基化,調(diào)節(jié)染色質(zhì)結(jié)構(gòu)或基因表達(dá),在炎癥和多種惡性腫瘤發(fā)生中發(fā)揮重要的表觀遺傳學(xué)作用。本課題組前期研究發(fā)現(xiàn),JMJD2B通過調(diào)控相關(guān)的細(xì)胞生物學(xué)過程在胃癌的發(fā)生發(fā)展過程中發(fā)揮重要的作用。然而,JMJD2B在胃癌細(xì)胞中高表達(dá)的機(jī)制尚不清楚。鑒于幽門螺桿菌感染是胃黏膜惡性轉(zhuǎn)化的重要始動(dòng)因素,因此,本研究探討了幽門螺桿菌對(duì)JMJD2B的調(diào)控作用及機(jī)制。另外,研究發(fā)現(xiàn)幽門螺桿菌感染可以上調(diào)細(xì)胞COX2的表達(dá),COX2在幽門螺桿菌致胃黏膜上皮細(xì)胞病變的過程中,不僅可以加重炎癥反應(yīng),還可促進(jìn)胃癌的發(fā)生發(fā)展。雖然已有研究揭示COX2在幽門螺桿菌致癌過程中的作用,但幽門螺桿菌激活COX2表達(dá)的分子機(jī)制仍未完全闡明。近期研究發(fā)現(xiàn)COX2的表達(dá)受到表觀遺傳學(xué)修飾的調(diào)控,如DNA甲基化、組蛋白乙�；�,與胃癌的進(jìn)展及預(yù)后密切相關(guān)。因此,本實(shí)驗(yàn)對(duì)JMJD2B介導(dǎo)的表觀遺傳學(xué)機(jī)制是否參與調(diào)控幽門螺桿菌誘導(dǎo)的COX2的表達(dá)上調(diào)進(jìn)行了研究。目的探討組蛋白去甲基化酶JMJD2B在幽門螺桿菌誘導(dǎo)的炎癥向胃癌惡性轉(zhuǎn)化過程中的作用及相關(guān)分子機(jī)制,進(jìn)一步闡明幽門螺桿菌的致癌機(jī)制。方法1.幽門螺桿菌感染調(diào)控JMJD2B表達(dá)的機(jī)制和功能研究：QRT-PCR、western blot和雙熒光素酶報(bào)告基因等方法檢測(cè)幽門螺桿菌感染對(duì)JMJD2B表達(dá)的調(diào)控,同時(shí)利用克隆形成實(shí)驗(yàn)分析JMJD2B對(duì)幽門螺桿菌誘導(dǎo)的細(xì)胞增殖的影響。利用β-catenin的小干擾RNA轉(zhuǎn)染至細(xì)胞,通過QRT-PCR和western blot檢測(cè)fMJD2B的表達(dá)。另外,構(gòu)建JMJD2B的啟動(dòng)子質(zhì)粒和突變質(zhì)粒,通過雙熒光素酶報(bào)告基因檢測(cè)β-catenin對(duì)JMJD2B表達(dá)的調(diào)控。2. JMJD2B在幽門螺桿菌誘導(dǎo)的COX2表達(dá)上調(diào)中的作用及其機(jī)制研究：將JMJD2B的小干擾RNA轉(zhuǎn)染至細(xì)胞,利用QRT-PCR. western blot和雙熒光素酶報(bào)告基因等方法檢測(cè)JMJD2B對(duì)COX2表達(dá)的調(diào)控。通過免疫共沉淀和染色質(zhì)免疫共沉淀實(shí)驗(yàn)研究JMJD2B調(diào)控COX2表達(dá)的分子機(jī)制。3.幽門螺桿菌調(diào)控JMJD2B和COX2表達(dá)的動(dòng)物實(shí)驗(yàn)：采用灌胃方式將幽門螺桿菌SS1注入C57BL/6J、鼠胃內(nèi)構(gòu)建模型,通過QRT-PCR檢測(cè)小鼠胃組織中的MJD2B和COX2表達(dá)水平。4. JMJD2B和COX2在幽門螺桿菌感染的胃組織標(biāo)本中的表達(dá)：收集臨床胃炎及胃癌患者的胃組織標(biāo)本,通過免疫組織化學(xué)方法檢測(cè)JMJD2B和COX2在正常組織、慢性非萎縮性胃炎、慢性萎縮性胃炎及胃癌組織標(biāo)本中的表達(dá)水平,確定JMJD2B和COX2在不同組織中的表達(dá)差異,并分析JMJD2B表達(dá)與幽門螺桿菌感染以及與COX2表達(dá)之間的相關(guān)性。結(jié)果1.幽門螺桿菌感染上調(diào)JMJD2 B表達(dá),這種調(diào)控作用不依賴其毒力因子CagA。2. β-catenin調(diào)控JMJD2B啟動(dòng)子活性介導(dǎo)幽門螺桿菌感染誘導(dǎo)的JMJD2B的轉(zhuǎn)錄表達(dá)。3. JMJD2B是幽門螺桿菌誘導(dǎo)的COX2表達(dá)上調(diào)中的關(guān)鍵調(diào)節(jié)分子。JMJD2B與NF-κB相互作用,共同結(jié)合于COX2啟動(dòng)子區(qū),使組蛋白H3K9me3發(fā)生去甲基化反應(yīng),轉(zhuǎn)錄激活COX2表達(dá)。4.動(dòng)物實(shí)驗(yàn)表明,幽門螺桿菌感染的鼠胃黏膜組織中JMJD2B和COX2的表達(dá)水平均升高。5.免疫組化結(jié)果表明,JMJD2B和COX2在正常組織、慢性非萎縮性胃炎、慢性萎縮性胃炎及胃癌組織標(biāo)本中的表達(dá)水平均逐漸升高,且JMJD2B的表達(dá)與幽門螺桿菌的感染狀態(tài)及COX2的表達(dá)均具有相關(guān)性。結(jié)論幽門螺桿菌感染胃黏膜上皮細(xì)胞誘導(dǎo)P-catenin移位入核,通過轉(zhuǎn)錄因子LEF/TCF結(jié)合至JMJD2B的啟動(dòng)子區(qū),上調(diào)JMJD2B的表達(dá)。JMJD2B結(jié)合于COX2啟動(dòng)子區(qū),使組蛋白H3K9me3發(fā)生去甲基化反應(yīng),改變?nèi)旧|(zhì)構(gòu)象,招募轉(zhuǎn)錄因子NF-κB結(jié)合至COX2的啟動(dòng)子區(qū),激活COX2的轉(zhuǎn)錄表達(dá),促進(jìn)了胃黏膜上皮細(xì)胞的惡性轉(zhuǎn)化。本研究從細(xì)胞水平、動(dòng)物水平和組織水平三方面探討了幽門螺桿菌感染調(diào)控JMJD2B表達(dá)促進(jìn)胃黏膜上皮細(xì)胞惡性轉(zhuǎn)化的相關(guān)分子機(jī)制,提示JMJD2B有可能作為一種新的胃癌治療的靶點(diǎn),有助于胃癌的早期診斷和干預(yù)。
[Abstract]:Background Gastric cancer is one of the most common malignancies worldwide and the mortality rate is the third in global cancer-related mortality. China is a high incidence of gastric cancer and the incidence of gastric cancer in China ranks second in the world after Japan. The occurrence of gastric cancer is the result of multifactorial co-action, in which Helicobacter pylori (H. pylori) infection is an important pathogenic factor of gastric cancer. Although there have been a large number of studies confirming the relationship between Helicobacter pylori infection and the occurrence of gastric cancer, the exact pathogenesis of Helicobacter pylori is still unclear. Recent studies have found that Helicobacter pylori infection can regulate the changes in epigenetic modification of host cells, such as changes in histones modification states, DNA methylation, and the like, which are closely related to the carcinogenic effects of H. pylori. Histone de-methyl parathion-2B, also known as KD4B, is one of the newly discovered HMGBJD2 protein family, which mainly targeted the 3-methyl (H3K9me3) of the 9-bit lysine of histones H3 to demethylation, regulate chromatin structure or gene expression, plays an important epigenetic role in the occurrence of inflammation and various malignant tumors. In the previous study, it was found that the cell biology process of Jurkat 2B played an important role in the development of gastric cancer. However, the mechanism of high expression of cyclin 2B in gastric cancer cells is unknown. Helicobacter pylori infection is an important starting factor in malignant transformation of gastric mucosa. In addition, it is found that Helicobacter pylori infection can upregulate the expression of COX2, and COX2 can not only aggravate inflammatory response, but also promote the development of gastric cancer. Although studies have revealed the role of COX2 in the carcinogenesis of Helicobacter pylori, the molecular mechanism of Helicobacter pylori activated COX2 expression has not been fully elucidated. Recently, it has been found that the expression of COX2 is regulated by epigenetic modification, such as DNA methylation, histones and so on. It is closely related to the progression and prognosis of gastric cancer. Therefore, this study conducted a study on whether the epigenetic mechanism mediated by HMGB2B was involved in the regulation of the expression of COX2 induced by H. pylori. Objective To investigate the role and molecular mechanism of histones to the malignant transformation of Helicobacter pylori induced by Helicobacter pylori and to further clarify the carcinogenic mechanism of H. pylori. Method 1. The mechanism and function of Helicobacter pylori infection regulating the expression of Helicobacter pylori 2B were studied: QRT-PCR, Western blot and double luciferase reporter gene. The expression of fMCS2B was detected by QRT-PCR and Western blot. In addition, the promoter plasmid and the mutant plasmid of Jurkat 2B were constructed, and the regulation of the expression of Jurkat 2B was detected by double luciferase reporter gene. The role of cyclin 2B in the upregulation of COX2 expression induced by Helicobacter pylori and its mechanism were studied: the small interfering RNA of Jurkat 2B was transfected into the cells and QRT-PCR was used. Western blot and luciferase reporter gene assay were used to detect the regulation of COX2 expression. The molecular mechanism of COX2 expression was studied by immune co-precipitation and chromatin immune co-precipitation experiment. The results showed that Helicobacter pylori SS1 was injected into C57BL/ 6J mice by gavage, and the expression levels of MCS2B and COX2 in gastric tissues of mice were detected by QRT-PCR. The expression of Jurkat 2B and COX2 in gastric tissue specimens of Helicobacter pylori infection: the collection of gastric tissue specimens of clinical gastritis and gastric cancer patients, the detection of COX2 in normal tissues and chronic non-atrophic gastritis by immunohistochemistry. The levels of expression in the specimens of chronic atrophic gastritis and gastric cancer were determined. The differences in the expression of cyclin 2B and COX2 in different tissues were determined, and the correlation between the expression of cyclin 2B and the infection of Helicobacter pylori and the expression of COX2 was analyzed. Result 1. Helicobacter pylori infection upregulated the expression of cyclin JD2 B, which did not depend on its virulence factor CagA. 2. Gene-catenin regulates the transcription and expression of cyclin 2B induced by Helicobacter pylori infection by the promoter activity of Jurkat 2B promoter. Jurkat 2B is a key regulatory molecule in the upregulation of COX2 expression induced by Helicobacter pylori. Jurkat 2B interacts with NF-Sepharose B and binds to the COX2 promoter region, so that the histones H3K9me3 are subjected to methylation reaction, and the transcription activates COX2 expression. Animal experiments showed that the expression levels of cyclin 2B and COX2 in gastric mucosa tissues of Helicobacter pylori infection increased. The results showed that the expression level of cyclin 2B and COX2 in normal tissues, chronic non-atrophic gastritis, chronic atrophic gastritis and gastric cancer tissue specimens increased gradually, and the expression of Jurkat 2B was correlated with the infection status of Helicobacter pylori and the expression of COX2. Conclusions Helicobacter pylori infection of gastric mucosa epithelial cells induces P-catenin migration into the nucleus, and the transcription factor LEF/ TCF binds to the promoter region of Jurkat 2B, and the expression of Jurkat 2B is up-regulated. Jurkat 2B binds to the COX2 promoter region to demethylation the histones H3K9me3, changes chromatin conformation, and recruits the transcription factor NF-5B to the promoter region of COX2, activates the transcription expression of COX2, and promotes the malignant transformation of gastric mucosa epithelial cells. In this study, we discussed the molecular mechanism of Helicobacter pylori infection regulated by Helicobacter pylori infection and promoted the malignant transformation of gastric epithelial cells from three aspects: cell level, animal level and tissue level. It is helpful for early diagnosis and intervention of 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】：2016
【分類號(hào)】：R735.2

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本文編號(hào)：2297758