乙型肝炎病毒X蛋白对血管内皮生长因子的调节通路研究

摘要

Objective To study the function of nuclear factor-kB(NF-kB) signaling pathway in regulating vascular endothelial growth factor (VEGF) by hepatitis B virus X protein (HBx). Methods After the establishment of LO2-HBx cell line with stable transfected HBx gene, NF-kB signaling pathway blocker PDTC was introduced to cut off its signal transduction. Double immunofluorescent staining and laser scanning confocal microscopy were applied to study the activation and deactivation of NF-kB signaling pathway. Real-time PCR and Western blot were used to observe the expression of VEGF gene before and after the HBx transfection, as well as the treatment with PDTC. Results The NF-kB signaling pathway of L02-HBx cells was activated after transfection with HBx gene as compared to the control L02 cells without treatment. The mRNA and protein levels of VEGF in L02-HBx cells increased 4. 07±0. 31 and 4. 34±0. 64 times respectively. The difference was of statistical significance ( P < 0. 05 ) in comparision with the control cells. The mRNA levels of VEGF decreased to 2. 33 ± 0. 22 and 1.86 ± 0. 18 ( P < 0. 05 ) and at the same time the expression of VEGF also reduced to 2. 52 ± 0. 29 and 2. 17 ± 0. 34 ( P < 0. 05 ), after treatment with 25.0 μmol/L and 50. 0 μmol/L PDTC for 24 h respectively when the NF-kB signaling pathway was blocked.There was no significant difference in VEGF mRNA and protein levels when treated with 12. 5 μmol/L PDTC for 24 h. Conclusion NF-kB signaling pathway maybe one of the routes through which HBx up-regulate the expression of VEGF to promote angiogenesis in hepatocellular carcinoma.%目的 研究NF-kB信号转导通路在乙型肝炎病毒X蛋白(HBx)调节血管内皮生长因子(VEGF)表达中的作用.方法 建立稳定转染HBx基因的L02细胞系(L02-HBx),用不同浓度的NF-kB信号转导通路阻断剂吡咯二硫氢基甲醋酯(PDTC)阻断NF-kB信号转导通路,荧光双标激光扫描共聚焦显微镜观察转染前后及PDTC加入前后NF-kB信号转导通路的激活及失活情况,同时用实时荧光定量PCR和Western blot技术检测转染前后及PDTC加入前后VEGF在mRNA及蛋白水平的表达变化.结果 以L02细胞为参照,转染HBx基因后的L02-HBx细胞NF-kB信号转导通路被激活,VEGF mRNA和蛋白水平分别增加(4.07±0.31)和(4.34±0.64)倍,差异有统计学意义(P<0.05);加入25.0和50.0μmol/L的PDTC作用24 h后,L02-HBx细胞NF-kB信号转导通路被阻断,VEGF mRNA表达水平分别增加(2.33±0.22)和(1.86±0.18)倍;VEGF蛋白表达水平分别增加(2.52±0.29)和(2.17±0.34)倍,与未加入PDTC的L02-HBx细胞的差异有统计学意义(P<0.05).12.5μmol/L PDTC作用24 h后,VEGF mRNA和蛋白水平的变化无统计学意义(P>0.05).结论 NF-kB信号转导通路是HBx上调VEGF表达、促进肝癌血管生成的途径之一.