上海交通大学学报（医学版）

›› 2012, Vol. 32 ›› Issue (3): 283-.doi: 10.3969/j.issn.1674-8115.2012.03.010

• 论著（基础研究） • 上一篇    下一篇

脂多糖对人胃癌细胞环氧化酶-2基因启动子活性的影响

徐 苓, 马妍慧, 袁向亮, 沈立松   

  1. 上海交通大学 医学院附属新华医院检验科, 上海 200092
  • 出版日期:2012-03-28 发布日期:2012-03-28
  • 通讯作者: 沈立松, 电子信箱: lisongshen@hotmail.com。
  • 作者简介:徐 苓（1985—）, 女, 检验师, 学士；电子信箱: immortality0018@yahoo.com.cn。

Effects of lipopolysaccharide on activity of COX-2 gene promoter of human gastric cancer cells

XU Ling, MA Yan-hui, YUAN Xiang-liang, SHEN Li-song   

  1. Department of Clinical Laboratory, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China
  • Online:2012-03-28 Published:2012-03-28

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摘要：

目的 研究炎症状态下人胃癌细胞中环氧化酶-2（COX-2）基因启动子活性。方法 采用免疫组织化学染色方法观察人胃癌组织中COX-2的表达情况。构建人COX-2基因启动子重组质粒pGL3-COX-2-promoter，采用脂质体介导转染法将重组质粒和内参质粒pRL-TK共转染胃癌细胞株SGC-7901和BGC-823。以不同质量浓度的脂多糖（LPS）工作液刺激细胞，于干预后不同时间点收集细胞，利用双荧光报告系统进行双荧光素酶报告基因活性检测，Western blotting检测COX-2蛋白的表达。结果 免疫组织化学染色观察发现，胃癌癌细胞胞质及癌旁黏膜上皮细胞的胞质中均有COX-2蛋白表达。在相同时间点，SGC-7901和BGC-823内COX-2基因启动子活性随LPS质量浓度的上升而增高（P<0.05）；高浓度（10 μg/mL）LPS刺激6 h后COX-2基因启动子活性达到峰值，随后逐渐降低 (P<0.05)。Western blotting检测结果显示：在10 μg/mL LPS干预下，SGC-7901和BGC-823内COX-2蛋白表达随LPS干预时间的延长而呈现上升趋势。结论 炎症刺激可激活人胃癌细胞COX-2基因启动子的表达活性。

关键词: 胃癌, 环氧化酶-2, 启动子, 双荧光素酶报告基因, 脂多糖

Abstract:

Objective To investigate the activity of cyclooxygenase-2 (COX-2) gene promoter of human gastric cancer cells under inflammatory state. Methods The expression of COX-2 in human gastric cancer tissues was detected with immunohistochemical staining. Recombinant pGL3-COX-2-promoter of human COX-2 gene promoter was constructed, and gastric cancer cell line SGC-7901 and BGC-823 were co-transfected with pGL3-COX-2-promoter and pRL-TK with lipofectin reagent-mediated transfection. Cells were collected after treatment with different mass concentrations of lipopolysaccharide (LPS) for different time, the activity of dual-luciferase reporter gene was determined with dualluciferase reporter system, and the expression of COX-2 protein was detected by Western blotting. Results Immunohistochemical staining indicated that COX-2 protein expressed both in cytoplasm of gastric cancer cells and cytoplasm of para-cancer mucosal epithelial cells. At the same time point, the activity of COX-2 gene promoter of SGC-7901 cells and BGC-823 cells increased with the mass concentrations of LPS （P<0.05）. The activity of COX-2 gene promoter reached the peak after treatment with high concentration of LPS (10 μg/mL) for 6 h, and then gradually decreased (P<0.05). Western blotting revealed that the expression of COX-2 protein of SGC-7901 cells and BGC-823 cells increased with the time of treatment with 10 μg/mL LPS. Conclusion Inflammatory stimulation may activate COX-2 gene promoter of human gastric cancer cells.

Key words: gastric cancer, cyclooxygenase-2, promoter, dual-luciferase report gene, lipopolysaccharide