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机构地区:[1]郑州大学基础医学院,河南省郑州市450001 [2]武警河南总队郑州市支队卫生队,河南省郑州市450006 [3]南阳市油田总医院,河南省南阳市473132
出 处:《世界华人消化杂志》2011年第18期1874-1879,共6页World Chinese Journal of Digestology
摘 要:目的:研究在人肝细胞L-02中乙型肝炎病毒X蛋白(HBx)对环氧化酶2(COX-2)表达的影响.方法:构建HBx基因表达载体pIRES2-AcGFP-HBx,转染入人肝细胞L-02中,RT-PCR和Westernblot检测HBx对COX-2表达的影响;通过细胞生长曲线和细胞周期变化,检测HBx蛋白对细胞分裂增殖的作用.同时将含有COX-2启动子核心片段的荧光素酶报告载体pGL3-COX-2转染入上述转染细胞,检测细胞荧光素酶荧光值以观察HBx蛋白对COX-2启动子活性的影响.结果:RT-PCR结果显示仅在HBx基因转染组细胞中有HBxmRNA的表达,且该组细胞中COX-2mRNA相对表达量显著高于空载体对照组和空白对照组(0.76±0.12vs0.28±0.04,0.25±0.03,均P<0.01);Westernblot结果显示,仅HBx基因转染组可见HBx蛋白有明显印迹条带,且该组细胞中COX-2蛋白免疫印迹较两对照组深;细胞生长曲线测定表明HBx基因转染组细胞在第3、4、5天的生长较两对照组显著加快(P<0.05);细胞周期测定显示与两对照组相比,HBx基因转染组G0-G1期比例显著降低,S期和G2-M期比例显著增加(P<0.05).HBx基因转染组COX-2启动子荧光素酶荧光值显著高于空载体对照组和空白对照组(1675.2±84.9vs657.7±34.7,739.3±45.3,均P<0.05).结论:HBx蛋白可增强人肝L-02细胞中COX-2的表达水平,对L-02细胞分裂增殖、生长有明显促进作用;HBx蛋白可通过提高COX-2启动子的活性以增强COX-2的表达.AIM: To investigate the effect of hepatitis B virus X protein (HBx) on COX-2 expression in human liver cell line L-02. METHODS: HBx expression vector pIRES2- AcGFP-HBx was constructed and transfected into L-02 cells. The expression of COX-2 mRNA and protein was detected by RT-PCR and West- ern blot, respectively. The effect of HBx protein on cell division and proliferation was evaluated by plotting cell growth curve and analyzing cell cycle. Moreover, pGL3-COX-2 plasmid, in which the COX-2 promoter has been linked to the luciferase reporter gene, was transfected into L-02 cells and luciferase activities were measured. RESULTS: RT-PCR results revealed that HBx mRNA was expressed only in cells transfected with the HBx gene, and that COX-2 mRNA expression in cells transfected with the HBx gene was higher than that in cells untranfected or transfected with an empty vector (0.76 ± 0.12 vs 0.28 ± 0.04, 0.25 ± 0.03, both P 〈 0.01). Western blot analysis showed that HBx protein was expressed only in cells transfected with the HBx gene, and COX-2 protein expression in this group was higher than that in the two control groups. The proliferation of cells transfected with the HBx gene was faster than that of control cells (both P 〈 0.05). The numbers of cells in S and G2-M phases significantly increased while those in G0-G1 phase decreased in cells transfected with the HBx gene compared to control cells (all P 〈 0.05). The luciferase activity in cells transfected with the HBx gene was higher than that in control cells (1 675.2 ± 84.9 vs 657.7 ± 34.7, 739.3 ±45.3, both P 〈 0.05). CONCLUSION: HBx protein can enhance COX-2 expression by up-regulating the activity of COX-2 promoter and promote cell growth, division and proliferation in human liver cell line L-02.
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