幽门螺杆菌相关胃癌基因差异表达谱的研究
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摘要
我国胃癌的发生率在各类恶性肿瘤中位居第二,是严重危害人民身体健康的疾病。20世纪80年代以来,在人群中具有较高感染率的幽门螺杆菌(Helicobacter pylori, Hp)与胃癌的关系已受到广泛关注。流行病学证实,Hp慢性感染是导致胃癌的重要危险因素之一,与胃癌的发生发展密切相关,感染人群患胃癌的风险比非感染者高4倍。大多数研究表明在“慢性胃炎—胃黏膜萎缩—肠化生—异型增生—胃癌”这一癌变模式中,Hp可能起着先导作用。1998年Watanabe等首次报道Hp感染的蒙古沙土鼠可发生胃癌,这是Hp感染可诱发胃癌的直接证据。1994年,世界卫生组织国际癌症研究机构(IARC)正式将Hp列为胃癌的I类致癌原。但Hp引起胃癌的机制尚未完全阐明。
胃癌的发生是一个多病因、多阶段的过程,感染Hp后,其结局可能发生慢性胃炎,也可能发展为胃癌。提示不同的菌株可能对胃黏膜上皮细胞产生不同的影响,导致不同程度的疾病发生。
本实验选用临床分离自胃癌、胃炎患者的菌株各10例分别和GES-1细胞以不同浓度比例共培养,12h、24h和48h后分别收获细胞,进行MTT和流式细胞仪检测。结果显示不同来源的Hp菌株对GES-1细胞在增殖方面的影响具显著性差异,低浓度菌液可不同程度地促进细胞增殖,高浓度菌液则抑制细胞增殖活性,并且随着时间延长,低浓度菌液促细胞增殖作用逐渐增强,高浓度菌液对细胞增殖的抑制作用也越发明显。不同疾病来源的Hp菌株均会引起胃黏膜上皮细胞凋亡率增加,但组间差异无显著性。随着时间的延长和细菌浓度的提高,Hp菌株促胃黏膜上皮细胞凋亡的影响越发显著。
分别挑选出对GES-1细胞的增殖影响最显著和最小的两株Hp菌株,分别和GES-1细胞共培养,收获细胞后提取RNA产物与全基因组表达谱芯片杂交后进行统计分析。结果显示Hp感染GES-1细胞后引起胃黏膜上皮细胞基因表达谱广泛地改变,这些差异表达基因与原癌基因与抑癌基因的激活或失活、细胞周期、细胞骨架和运动、细胞凋亡、DNA的合成、转录、细胞信号、蛋白质的翻译、物质和能量代谢等过程有关。筛选出一些与肿瘤的发生发展密切相关的基因,如TRAF1、HDAC6、ETS1、JUN、JUNB、CLDN1、CXCL2、IL-8、IRAK2、NUPR1、STC2、TP53BP1、TNFAIP3、DIO2以及SR蛋白家族,为下一步的基因表达验证和生物学功能研究提供了新的线索。
在基因芯片的结果中挑选出差异性较大且科研意义较高、目前在Hp相关性胃癌发病机制尚未见报道的两个基因：TRAF1和HDAC6。将挑选出的对GES-1细胞的增殖影响最显著和最小的两株Hp菌株,分别和GES-1细胞共培养,用空白组进行对照,共分为对照组、胃炎组和胃癌组,共培养24小时后。收获细胞提取RNA产物,逆转录后得到cDNA,进行RT-PCR检测,结果发现TRAF1和HDAC6在对照组、胃炎组、胃癌组中的表达依次增强,与基因芯片结果一致。
利用取自于临床的伴Hp感染的慢性浅表性胃炎、萎缩性胃炎(病检示肠上皮化生、不典型增生)及胃癌患者的标本进行免疫组化检测。结果显示TRAF1和HDAC6在慢性浅表性胃炎、肠上皮化生、不典型增生及胃癌组织中表达依次增强；提示TRAF1和HDAC6可能参与了Hp感染导致慢性浅表性胃炎、萎缩性胃炎、胃癌的发生发展过程,为阐明Hp致胃癌发生的分子机制以及胃癌新的分子标记物提供了新的线索。
The incidence of gastric cancer is in the second place in various cancers in China. It is a serious harm to the health of people. Since 80s of the 20th century, the relation of gastric cancer and H.pylori infection has received extensive attention. Epidemiology confirmed that chronic infection of H.pylori was a important risk factors for gastric cancer, and the risk of infected people was 4 times higher than non-infected. Many researches showed that there was the mode of "chronic gastritis-gastric atrophy-intestinal metaplasia-dysplasia-gastric cancer" in which H.pylori played a leading role. In 1998, Watanabe first reported that Mongolian gerbil infected by H.pylori could causegastric cancer. In 1994, IARC of the World Health Organization made H.pylori as a classⅠcarcinogen. But the mechanism of gastric cancer caused by H.pylori has not been fully understood.
The carcinogenesis of gastric cancer is a multi-cause, multi-stage process. Infected by H.pylori, it may lead to chronic gastritis or gastric cancer. It was indicated that different strains may have different effects on gastric epithelial cells and cause different diseases.
The experiment used 20 strains respectively isolated from the patients with gastric cancer and gastritis. The strains were co-cultured in different concentration ratio with GES-1 cells. The cells were collected after 12,24 and 48 hours and followed by MTT and flow cytometry. The result showed that H.pylori strains from different sources had a significant difference on the proliferation of GES-1 cells. Low concentrations of H.pylori can promote the proliferation of cells in different degrees whereas high concentrations can inhibit the proliferation. The activity of promoting of low concentrations in proliferation increased gradually by time, so as the inhibiting of high concentrations. H.pylori strains from different sources can induce the apoptosis of gastric epithelial cells with no significant difference. As the concentrations and time increased, H.pylori strains had more significant effect on the apoptosis of gastric epithelial cells.
The two H.pylori strains, which had the most significant and least effect on the proliferation of GES-1 cells, were selected infect Mongolian gerbils.and co-cultured with GES-1 cells. The tRNA products were extracted after the cells were collected, and then the whole-genome microarray hybridization were analyzed. The results showed that GES-1 cells infected by H.pylori can cause extensive changes on gene expression of gastric epithelial cells. These differentially expressed genes were related with activation and inactivation of proto-oncogenes and tumor suppressor gene, cell cycle, movement of cytoskeleton, apoptosis, DNA synthesis, transcription, cell signaling, protein translation, material and energy metabolism and so on. Some genes closely related with cancer, such as ETS1、JUN、JUNB、CLDN1、CXCL2、IRAK2、NUPR1、STC2、TP53BP1、TNFAIP3、TRAF1、DIO2、ST13 and SR protein family were filtered out for further investigation of gene expression and biological function.
Among 2834 and 314 genes that changed expression pattern after infection of the two strains, we validated HDAC6 (histone deacetylase 6) and TRAF1 (tumor necrosis factor receptor-associated factor 1) genes,both of which showed elevated expression level compared to the control. HDAC6 is implicated in tumor growth in that it contributes to oncogenic cell transformation. HDAC inhibitors have been proved to have antitumor activity in hematologic and solid tumors by both preclinical studies and clinical trials. TRAF1 can form a heterodimeric complex with TRAF2, which is required for TNF-alpha-mediated activation of MAPK8/JNK and NF-kappaB. Recent research showed that TRAF1 is over expressed in malignant cells. To the best of our knowledge, this is the first report showing that HDAC6 and TRAF1 highly expressed in H.pylori infected cells. Our research result provided new clues for the pathogenic mechanism of H.pylori.
胃癌的发生是一个多病因、多阶段的过程,感染Hp后,其结局可能发生慢性胃炎,也可能发展为胃癌。提示不同的菌株可能对胃黏膜上皮细胞产生不同的影响,导致不同程度的疾病发生。
本实验选用临床分离自胃癌、胃炎患者的菌株各10例分别和GES-1细胞以不同浓度比例共培养,12h、24h和48h后分别收获细胞,进行MTT和流式细胞仪检测。结果显示不同来源的Hp菌株对GES-1细胞在增殖方面的影响具显著性差异,低浓度菌液可不同程度地促进细胞增殖,高浓度菌液则抑制细胞增殖活性,并且随着时间延长,低浓度菌液促细胞增殖作用逐渐增强,高浓度菌液对细胞增殖的抑制作用也越发明显。不同疾病来源的Hp菌株均会引起胃黏膜上皮细胞凋亡率增加,但组间差异无显著性。随着时间的延长和细菌浓度的提高,Hp菌株促胃黏膜上皮细胞凋亡的影响越发显著。
分别挑选出对GES-1细胞的增殖影响最显著和最小的两株Hp菌株,分别和GES-1细胞共培养,收获细胞后提取RNA产物与全基因组表达谱芯片杂交后进行统计分析。结果显示Hp感染GES-1细胞后引起胃黏膜上皮细胞基因表达谱广泛地改变,这些差异表达基因与原癌基因与抑癌基因的激活或失活、细胞周期、细胞骨架和运动、细胞凋亡、DNA的合成、转录、细胞信号、蛋白质的翻译、物质和能量代谢等过程有关。筛选出一些与肿瘤的发生发展密切相关的基因,如TRAF1、HDAC6、ETS1、JUN、JUNB、CLDN1、CXCL2、IL-8、IRAK2、NUPR1、STC2、TP53BP1、TNFAIP3、DIO2以及SR蛋白家族,为下一步的基因表达验证和生物学功能研究提供了新的线索。
在基因芯片的结果中挑选出差异性较大且科研意义较高、目前在Hp相关性胃癌发病机制尚未见报道的两个基因：TRAF1和HDAC6。将挑选出的对GES-1细胞的增殖影响最显著和最小的两株Hp菌株,分别和GES-1细胞共培养,用空白组进行对照,共分为对照组、胃炎组和胃癌组,共培养24小时后。收获细胞提取RNA产物,逆转录后得到cDNA,进行RT-PCR检测,结果发现TRAF1和HDAC6在对照组、胃炎组、胃癌组中的表达依次增强,与基因芯片结果一致。
利用取自于临床的伴Hp感染的慢性浅表性胃炎、萎缩性胃炎(病检示肠上皮化生、不典型增生)及胃癌患者的标本进行免疫组化检测。结果显示TRAF1和HDAC6在慢性浅表性胃炎、肠上皮化生、不典型增生及胃癌组织中表达依次增强；提示TRAF1和HDAC6可能参与了Hp感染导致慢性浅表性胃炎、萎缩性胃炎、胃癌的发生发展过程,为阐明Hp致胃癌发生的分子机制以及胃癌新的分子标记物提供了新的线索。
The incidence of gastric cancer is in the second place in various cancers in China. It is a serious harm to the health of people. Since 80s of the 20th century, the relation of gastric cancer and H.pylori infection has received extensive attention. Epidemiology confirmed that chronic infection of H.pylori was a important risk factors for gastric cancer, and the risk of infected people was 4 times higher than non-infected. Many researches showed that there was the mode of "chronic gastritis-gastric atrophy-intestinal metaplasia-dysplasia-gastric cancer" in which H.pylori played a leading role. In 1998, Watanabe first reported that Mongolian gerbil infected by H.pylori could causegastric cancer. In 1994, IARC of the World Health Organization made H.pylori as a classⅠcarcinogen. But the mechanism of gastric cancer caused by H.pylori has not been fully understood.
The carcinogenesis of gastric cancer is a multi-cause, multi-stage process. Infected by H.pylori, it may lead to chronic gastritis or gastric cancer. It was indicated that different strains may have different effects on gastric epithelial cells and cause different diseases.
The experiment used 20 strains respectively isolated from the patients with gastric cancer and gastritis. The strains were co-cultured in different concentration ratio with GES-1 cells. The cells were collected after 12,24 and 48 hours and followed by MTT and flow cytometry. The result showed that H.pylori strains from different sources had a significant difference on the proliferation of GES-1 cells. Low concentrations of H.pylori can promote the proliferation of cells in different degrees whereas high concentrations can inhibit the proliferation. The activity of promoting of low concentrations in proliferation increased gradually by time, so as the inhibiting of high concentrations. H.pylori strains from different sources can induce the apoptosis of gastric epithelial cells with no significant difference. As the concentrations and time increased, H.pylori strains had more significant effect on the apoptosis of gastric epithelial cells.
The two H.pylori strains, which had the most significant and least effect on the proliferation of GES-1 cells, were selected infect Mongolian gerbils.and co-cultured with GES-1 cells. The tRNA products were extracted after the cells were collected, and then the whole-genome microarray hybridization were analyzed. The results showed that GES-1 cells infected by H.pylori can cause extensive changes on gene expression of gastric epithelial cells. These differentially expressed genes were related with activation and inactivation of proto-oncogenes and tumor suppressor gene, cell cycle, movement of cytoskeleton, apoptosis, DNA synthesis, transcription, cell signaling, protein translation, material and energy metabolism and so on. Some genes closely related with cancer, such as ETS1、JUN、JUNB、CLDN1、CXCL2、IRAK2、NUPR1、STC2、TP53BP1、TNFAIP3、TRAF1、DIO2、ST13 and SR protein family were filtered out for further investigation of gene expression and biological function.
Among 2834 and 314 genes that changed expression pattern after infection of the two strains, we validated HDAC6 (histone deacetylase 6) and TRAF1 (tumor necrosis factor receptor-associated factor 1) genes,both of which showed elevated expression level compared to the control. HDAC6 is implicated in tumor growth in that it contributes to oncogenic cell transformation. HDAC inhibitors have been proved to have antitumor activity in hematologic and solid tumors by both preclinical studies and clinical trials. TRAF1 can form a heterodimeric complex with TRAF2, which is required for TNF-alpha-mediated activation of MAPK8/JNK and NF-kappaB. Recent research showed that TRAF1 is over expressed in malignant cells. To the best of our knowledge, this is the first report showing that HDAC6 and TRAF1 highly expressed in H.pylori infected cells. Our research result provided new clues for the pathogenic mechanism of H.pylori.
引文
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