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目的:探讨乙肝病毒X蛋白(HBx)对人正常肝细胞中生物钟基因表达的影响。方法:分别将HBx表达质粒(pcDNA3.1-HBx)与空载体质粒(pcDNA3.1)转染入人正常肝细胞系L02后,用RT-PCR与Western blot法检测细胞HBx mRNA与蛋白的表达;real-time PCR检测细胞中生物钟基因CLOCK、BMAL1、Per1、Per2、Per3、Cry1、Cry2、CKIε的表达。结果:RT-PCR与Western blot结果显示,L02细胞转染HBx表达质粒后有明显的HBx mRNA与蛋白表达,而转染空载体质粒的L02细胞无HBx mRNA与蛋白表达。与转染空载体质粒的L02细胞比较,转染HBx表达质粒的L02细胞CLOCK与Cry1的mRNA表达明显升高,而BMAL1、Per1、Per2、Per3、Cry2、CKIε的mRNA表达明显降低,差异均有统计学意义(均P<0.05)。结论:HBx能使正常肝细胞中的生物钟基因表达发生改变,破坏肝细胞正常的生物节律,这可能为其导致肝癌形成的机制之一。
Objective: To investigate the effect of hepatitis B virus X protein (HBx) on the gene expression of circadian clock in human normal liver cells. Methods: HBx expression plasmid (pcDNA3.1-HBx) and empty vector plasmid (pcDNA3.1) were transfected into human normal liver cell line L02, and the expression of HBx mRNA and protein was detected by RT-PCR and Western blot Real-time PCR was used to detect the expression of the clock genes CLOCK, BMAL1, Per1, Per2, Per3, Cry1, Cry2 and CKIε in the cells. Results: RT-PCR and Western blot showed that HBx mRNA and protein expression were significantly increased in L02 cells transfected with HBx expression plasmid, but no expression of HBx mRNA and protein in L02 cells transfected with empty vector plasmid. Compared with L02 cells transfected with empty vector plasmid, the mRNA expression of CLOCK and Cry1 in L02 cells transfected with HBx expression plasmid was significantly increased, while the mRNA expression of BMAL1, Per1, Per2, Per3, Cry2 and CKIε was significantly decreased Statistical significance (all P <0.05). Conclusion: HBx can change the expression of circadian clock gene in normal liver cells and destroy the normal biological rhythm of hepatocytes, which may be one of the mechanisms that lead to the formation of liver cancer.