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目的:通过观察常氧与低氧下CagA+幽门螺杆菌(Hp)对人胃癌SGC7901细胞系HIF-2α和Oct-4表达的影响,初步探讨Hp在胃癌发生发展中的干细胞机制,及其与肿瘤微环境低氧的协同作用。方法:内镜下采集胃黏膜标本后行Hp培养并鉴定,PCR方法鉴定CagA基因。CagA+Hp与胃癌SGC7901细胞于常氧和低氧环境下共培养48h(分常氧对照组、低氧对照组、常氧CagA+Hp组、低氧CagA+Hp组)。免疫细胞化学法检测HIF-2α和Oct-4蛋白的表达,RT-PCR法检测Oct-4mRNA的表达。结果:免疫细胞化学结果显示,常氧下胃癌SGC7901细胞HIF-2α和Oct-4蛋白呈低水平表达,表达量分别为0.263 9±0.008 0和0.317 9±0.001 3。低氧和CagA+Hp均能显著诱导HIF-2α和Oct-4蛋白表达,与常氧对照组的相比,HIF-2α表达量为0.431 8±0.001 2和0.422 5±0.017 0,t值分别为46.410 3和18.875 6,P<0.01;Oct-4为0.546 8±0.008 2和0.478 9±0.001 8,t值分别为61.649 1和162.139 0,P<0.01。与低氧对照组和常氧CagA+Hp组相比,低氧CagA+Hp组蛋白表达量进一步升高,HIF-2α为0.507 7±0.007 0,t值分别为23.896 8和10.362 5,P<0.01;Oct-4为0.698 7±0.005 1,t值分别为35.173 7和90.876 1,P<0.01。相关分析显示,HIF-2α与Oct-4表达呈正相关,r=0.964 0,P<0.05。RT-PCR检测各组Oct-4mRNA表达结果显示与免疫细胞化学结果一致。结论:常氧下CagA+Hp可刺激胃癌细胞HIF-2α和Oct-4表达,低氧环境下其表达进一步增加,表明CagA+Hp和低氧对HIF-2α和Oct-4的表达有协同作用。提示CagA+Hp和低氧可能是诱导胃癌细胞干细胞化的重要原因。
OBJECTIVE: To investigate the effect of Hp and CagA + Helicobacter pylori (Hp) on the expression of HIF-2α and Oct-4 in human gastric cancer cell line SGC7901 under normoxia and hypoxia, and to explore the mechanism of Hp in the development of gastric cancer and its relationship with tumor Synergistic effect of micro-environment and hypoxia. Methods: Gastric mucosa specimens were collected by endoscopy and identified by Hp culture. The CagA gene was identified by PCR. CagA + Hp and gastric cancer SGC7901 cells were co-cultured for 48h (normoxia control group, hypoxia control group, normoxia CagA + Hp group, hypoxia CagA + Hp group) under normoxia and hypoxia conditions. The expression of HIF-2αand Oct-4protein were detected by immunocytochemistry, and the expression of Oct-4mRNA was detected by RT-PCR. Results: The results of immunocytochemistry showed that the expressions of HIF-2α and Oct-4 protein in gastric cancer cell line SGC7901 under normoxia were low, and the expression levels were 0.263 9 ± 0.008 0 and 0.317 9 ± 0.001 3 respectively. Hypoxia and CagA + Hp could significantly induce the expression of HIF-2α and Oct-4 protein. Compared with normoxic control group, the expression of HIF-2α was 0.431 8 ± 0.001 2 and 0.422 5 ± 0.017 0, respectively P <0.01; Oct-4 was 0.546 8 ± 0.008 2 and 0.478 9 ± 0.001 8, respectively; t values were 61.649 1 and 162.139 0 respectively, P <0.01. Compared with hypoxic control group and normoxia CagA + Hp group, the expression of Hypoxia CagA + Hp group protein increased further, HIF-2α was 0.507 7 ± 0.007 0, t values were 23.896 8 and 10.362 5 respectively, P < 0.01; Oct-4 was 0.698 7 ± 0.005 1, t values were 35.173 7 and 90.876 1 respectively, P <0.01. Correlation analysis showed that there was a positive correlation between the expression of HIF-2α and Oct-4, r = 0.964 0, P <0.05. RT-PCR detection Oct-4mRNA expression in each group showed the same results with the immunocytochemistry. CONCLUSION: CagA + Hp can stimulate the expression of HIF-2α and Oct-4 in gastric cancer cells under normoxia and increase in hypoxia environment, suggesting that CagA + Hp and hypoxia have a synergistic effect on the expression of HIF-2α and Oct-4 . It is suggested that CagA + Hp and hypoxia may be important factors in inducing gastric cancer cell stem cell.