幽门螺杆菌对GES-1细胞GATA3、Cx32、Cx43表达的影响及其相关性分析

发布时间：2018-07-05 11:44

本文选题：幽门螺杆菌 + GATA3　；参考：《中南大学》2014年硕士论文

【摘要】：目的：本课题组前期研究发现幽门螺杆菌(Helicobacter pylori, H.pylori)感染胃“炎-癌链”不同阶段(慢性非萎缩性胃炎→慢性萎缩性胃炎→肠上皮化生→异型增生→胃癌)胃黏膜组织的Cx32、Cx43表达逐渐降低,而GATA3表达逐渐升高,生物信息软件分析发现Cx32、Cx43启动子区域存在多个GATA3结合部位,提示GATA3可能调控Cx32、Cx43表达。本课题拟通过细胞模型,观察H.pylori对GES-1细胞GATA3表达的影响及其与Cx32、Cx43表达的关系,探讨H.pylori感染是否通过GATA3改变导致Cx32、Cx43表达降低而与胃癌发生有关。方法：实验组将临床分离自胃癌病人的东亚型CagA+H.pylori菌株与GES-1细胞按50:1共培养12h,24h,对照组不加H.pylori培养12h、24h。采用Real-time PCR及免疫印迹法(Western-blotting)检测GES-1细胞GATA3、Cx32、Cx43mRNA和蛋白质表达；划痕标记荧光染料示踪技术(SLDT)检测GES-1细胞GJIC功能。同时采用阳离子脂质体法转染GATA3siRNA序列至前期筛选出GATA3表达量最高的人胃癌细胞株BGC803,检测GATA3siRNA干扰效果及转染后Cx32、Cx43表达的变化。结果：(1)GATA3表达：实验组H.pylori感染GES-1细胞GATA3表达在转录及蛋白水平随着培养时间延长有升高趋势,24h表达量较12h及对照组24h均升高,差异有统计学意义(P0.05)。(2) Cx32、 Cx43表达：实验组H.pylori感染后GES-1细胞Cx32、Cx43表达在转录及蛋白水平均有下降趋势,24h表达量较12h及对照组24h均降低,差异有统计学意义(P0.05)。(3) GJIC功能改变：对照组GES-1细胞荧光染料向邻近细胞传递3-4列,具有较强的GJIC功能；实验组荧光染料大多局限于划痕旁的单列细胞,仅极少数传递1-2列,GJIC功能明显减弱或缺失。(4)GATA3与Cx32、Cx43表达的关系：H.pylori感染GES-1细胞后GATA3与Cx32、Cx43在转录及蛋白水平表达均呈负相关(P0.05); BGC803细胞转染GATA3siRNA后GATA3与Cx32、Cx43在转录及蛋白水平表达均呈负相关(P0.05)。结论：1、H.pylori上调GES-1细胞GATA3表达,同时下调Cx32、 Cx43表达,降低细胞GJIC功能。2、GATA3与Cx32、Cx43表达呈负相关,其可能靶向调节Cx32、Cx43,在H.pylori感染所致的胃癌中发挥重要作用。
[Abstract]:Objective: our previous study found that Helicobacter pylori (H.pylori) infection of gastric "inflammation and cancer chain" (chronic non-atrophic gastritis / chronic atrophic gastritis) gastric mucosa group The expression of Cx32Cx43 decreased gradually. However, the expression of GATA3 increased gradually, and the analysis of bioinformatics software showed that there were many GATA3 binding sites in the Cx32Cx43 promoter, suggesting that GATA3 might regulate the expression of Cx32Cx43. The purpose of this study was to observe the effect of H.pylori on the expression of GATA3 in GES-1 cells and its relationship with the expression of Cx32Cx43, and to explore whether H.pylori infection may lead to the decrease of Cx32Cx43 expression through the change of GATA3, which may be related to the occurrence of gastric cancer. Methods: East-Asian CagA H.pylori strains isolated from gastric cancer patients were co-cultured with GES-1 cells for 12 h or 24 h at 50:1 in the experimental group, while those in the control group were not cultured with H.pylori for 12 h or 24 h. Real-time PCR and Western-blotting were used to detect the expression of GATA3Cx32Cx43 mRNA and protein in GES-1 cells, and the GJIC function of GES-1 cells was detected by scratch-labeled fluorescent dye tracing (SLDT). At the same time, GATA3siRNA sequence was transfected with cationic liposome to screen the highest expression of GATA3 in human gastric cancer cell line BGC803. The interference effect of GATA3siRNA and the change of Cx32mCx43 expression after transfection were detected. Results: (1) GATA3 expression: the expression of GATA3 in GES-1 cells of H.pylori infection in experimental group increased with the increase of culture time. The expression of GATA3 at 24 h was higher than that in 12 h and control group. The difference was statistically significant (P0.05). (2) Cx32, Cx43 expression: after H.pylori infection, the expression of Cx32 Cx43 in GES-1 cells decreased in both transcription and protein levels. The expression of Cx32 and Cx43 in GES-1 cells decreased at 24 h after H.pylori infection, compared with 12 h and 24 h in control group. The difference was statistically significant (P0.05). (3): the fluorescent dyes of GES-1 cells in the control group were 3 to 4 rows, which had strong GJIC function, and the fluorescent dyes in the experimental group were mostly confined to the single cells next to the scratch. (4) the relationship between GATA3 and Cx32Cx43 expression in GES-1 cells infected with H. pylori was negatively correlated with the expression of GATA3 and Cx32Cx43 at transcription and protein levels (P0.05), and GATA3 and Cx32Cx43 in transcription and protein water after transfection of GATA3siRNA by BGC803 cells. The flat expression was negatively correlated (P0.05). Conclusion H. pylori can up-regulate GATA3 expression in GES-1 cells, down-regulate the expression of Cx32 and Cx43, and down-regulate the expression of Cx32 and Cx43, which may play an important role in H. pylori induced gastric cancer.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R573

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