本文選題：幽門螺桿菌 + GATA3��； 參考：《中南大學(xué)》2014年碩士論文

【摘要】：目的：本課題組前期研究發(fā)現(xiàn)幽門螺桿菌(Helicobacter pylori, H.pylori)感染胃“炎-癌鏈”不同階段(慢性非萎縮性胃炎→慢性萎縮性胃炎→腸上皮化生→異型增生→胃癌)胃黏膜組織的Cx32、Cx43表達(dá)逐漸降低,而GATA3表達(dá)逐漸升高,生物信息軟件分析發(fā)現(xiàn)Cx32、Cx43啟動(dòng)子區(qū)域存在多個(gè)GATA3結(jié)合部位,提示GATA3可能調(diào)控Cx32、Cx43表達(dá)。本課題擬通過細(xì)胞模型,觀察H.pylori對(duì)GES-1細(xì)胞GATA3表達(dá)的影響及其與Cx32、Cx43表達(dá)的關(guān)系,探討H.pylori感染是否通過GATA3改變導(dǎo)致Cx32、Cx43表達(dá)降低而與胃癌發(fā)生有關(guān)。 方法：實(shí)驗(yàn)組將臨床分離自胃癌病人的東亞型CagA+H.pylori菌株與GES-1細(xì)胞按50:1共培養(yǎng)12h,24h,對(duì)照組不加H.pylori培養(yǎng)12h、24h。采用Real-time PCR及免疫印跡法(Western-blotting)檢測GES-1細(xì)胞GATA3、Cx32、Cx43mRNA和蛋白質(zhì)表達(dá)；劃痕標(biāo)記熒光染料示蹤技術(shù)(SLDT)檢測GES-1細(xì)胞GJIC功能。同時(shí)采用陽離子脂質(zhì)體法轉(zhuǎn)染GATA3siRNA序列至前期篩選出GATA3表達(dá)量最高的人胃癌細(xì)胞株BGC803,檢測GATA3siRNA干擾效果及轉(zhuǎn)染后Cx32、Cx43表達(dá)的變化。 結(jié)果：(1)GATA3表達(dá)：實(shí)驗(yàn)組H.pylori感染GES-1細(xì)胞GATA3表達(dá)在轉(zhuǎn)錄及蛋白水平隨著培養(yǎng)時(shí)間延長有升高趨勢,24h表達(dá)量較12h及對(duì)照組24h均升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。(2) Cx32、 Cx43表達(dá)：實(shí)驗(yàn)組H.pylori感染后GES-1細(xì)胞Cx32、Cx43表達(dá)在轉(zhuǎn)錄及蛋白水平均有下降趨勢,24h表達(dá)量較12h及對(duì)照組24h均降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。(3) GJIC功能改變：對(duì)照組GES-1細(xì)胞熒光染料向鄰近細(xì)胞傳遞3-4列,具有較強(qiáng)的GJIC功能；實(shí)驗(yàn)組熒光染料大多局限于劃痕旁的單列細(xì)胞,僅極少數(shù)傳遞1-2列,GJIC功能明顯減弱或缺失。(4)GATA3與Cx32、Cx43表達(dá)的關(guān)系：H.pylori感染GES-1細(xì)胞后GATA3與Cx32、Cx43在轉(zhuǎn)錄及蛋白水平表達(dá)均呈負(fù)相關(guān)(P0.05); BGC803細(xì)胞轉(zhuǎn)染GATA3siRNA后GATA3與Cx32、Cx43在轉(zhuǎn)錄及蛋白水平表達(dá)均呈負(fù)相關(guān)(P0.05)。 結(jié)論：1、H.pylori上調(diào)GES-1細(xì)胞GATA3表達(dá),同時(shí)下調(diào)Cx32、 Cx43表達(dá),降低細(xì)胞GJIC功能。2、GATA3與Cx32、Cx43表達(dá)呈負(fù)相關(guān),其可能靶向調(diào)節(jié)Cx32、Cx43,在H.pylori感染所致的胃癌中發(fā)揮重要作用。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R573

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