摘要目的 研究硫酸类肝素-3-O-磺基转移酶B1(HS3ST381)对HBV复制的影响.方法 以HepG2细胞为阴性对照组,转染2.5μg pCH9-HBV(HBV表达质粒)的HepG2细胞为阳性对照组;转染了2.5μg pCH9-HBV、1.5 μg pcDNA3.1(HS3ST3B1表达质粒载体)和2.0μgpTZU6+1(干扰质粒构建载体)的HepG2细胞为对照组;转染了2.5μg pCH9-HBV、1.5 μgpCDNA3.1-HS3ST381和2.0 μg pTZU6+1的HepG2细胞为实验组A;转染了2.5 μg pCH9-HBV、1.5 μg pCDNA3.1-HS3ST381和2.0μg psh1126(HS3ST381的干扰质粒)的HepG2细胞为干扰组A.转染了2.5μg pCH9-HBV和2.0 μg pTZU6+1的HepG2细胞为实验组B;转染了2.5μgpCH9-HBV和2.0μg psh1126的HepG2为干扰组B.采用Southern blot和实时聚合酶链式反应技术检测HS3ST381共转染处理及未共转染处理的细胞内HBV复制中间体含量和病毒总RNA表达量,用双荧光素酶报告系统检测这种变化与HBV启动子[核心启动子(cp)、X蛋白启动子(xp)、包膜蛋白启动子1(sp1)、包膜蛋白启动子2(sp2)]活性的关系.采用SPSS17.0统计软件进行单因素方差分析,P<0.05为差异有统计学意义.结果以对照组HBV DNA含量为1,实验组A、干扰组A HBV DNA含量分别为10%±2%、31%±4%,对照组与实验组A比较,F=20.8,P=0.034,差异有统计学意义.实验组A与干扰组A比较,F=24.9,P=0.021,差异有统计学意义.与干扰组B比较,试验组B HBV DNA水平上升了130%±11%.在HBV稳定表达细胞株中转染pCDNA3.1-HS3ST381分别为0.5、1.0、1.5μg时,HBV DNA水平分别是对照组的90.0%±3.1%、82.0%±2.3%、21.0%±1.9%,与对照组比较,F值分别为22.7、20.3、26.5,P值分别为0.029、0.041、0.015,差异均有统计学意义.实验组A HBV总RNA量为对照组总RNA量的17.0%±2.7%,两组比较,F=25.6,P=0.018,差异有统计学意义.干扰组A HBV总RNA的量恢复到对照组的74.0%±3.9%,实验组A与干扰组A比较,F=21.3,P=0.032,差异有统计学意义.但HS3ST381对总RNA的下调与HBV的启动子活性无关.结论HS3ST381对HBV复制和HBV总RNA水平均有下调作用,但总RNA的下调不是HS3ST381直接作用于HBV启动子的结果.

abstractsObjective To investigate the effect of HS3ST3B1 on hepatitis B virus(HBV)replicaction.Methods HepG2 cells were classified into 7 groups according to the plasmids transfected: (1) Blank group,no plasmid transfected;2. Positive control,transfected with pCH9-HBV which permits HBV replication;(3)Negative control,transfected with pCH9-HBV + pcDNA3.1 + pTZU6+ 1;(4) Treatment A,transfected withpCH9-HBV + pCDNA3.1-HS3ST3B1 + pTZU6+1;(5) Interference A,transfected with pCH9-HBV +pCDNA3.1-HS3ST3B1 + psh1126 (a plasmid to interfere HS3ST3B1 expression);(6) Treatment B,transfected with pCH9-HBV + pTZU6+1;(7) Interference B,transfected with pCH9-HBV + psh1126. The levels of HBV DNA were detected in the above groups by Southern blotting. HBV total RNA of Negative control,Treatment A and Interference A were quantified by Real-time PCR to determine the influence of HS3ST3B1 over-expression on the HBV RNA transcription. The acitivitiy of the four HBV promoters[core promoter (cp),x promoter(xp),surface antigen promoter1(sp1),surface antigen promoter2 (sp2)]were assayed by Dual-Luciferase Reporter Assay System. The data was analyzed using one way ANOVA,with P < 0.05 indicaring statistically meaningful difference. Result Southern blot data revealed the level of HBV DNA in Treatment A and Interference A accounted for 10% ± 2% and 31% ± 4% of that in control. Compared with control,a statistical difference existed between Treatment A and Control,with F value equalling to 20.8 and P value equalling to 0.034 respectively. A statistical difference also existed between Interfere A and Treatment A,with F value equalling to 24.9 and P value eqalling to 0.021 respectively. The level of HBV DNA in Experiment B was raised by 130% ± 11% as compared to that in Interference B,and the levels of HBV DNA showed a dose-dependent decrease when H7 cells were transfected with 0.5,1.0,1.5μg pCDNA3.1-HS3ST3B1 respectively. Statistical differences existed between control and H7 transfected with different dose of pCDNA3.1-HS3ST3B1,with F values equalling to 22.7,20.3,26.5 and P values equalling to 0.029,0.041 and 0.015 respectively. Real-time PCR revealed that the HBV total RNA in Treatment A accounted for 17.0% ± 2.7%of that in control and there was a statistical difference between Treatment A and control,with F value equalling to 25.6 and P value equalling to 0.018. In addtion,HBV DNA in Interference A was restored to 74.0% ±3.9% of that in control,and there was also a statistical difference between Treatment A and Interference A,with F value equalling to 21.3 and P value equalling to 0.032. However,the downregulaiton of HBV total RNA had nothing to do with HBV promoters activity. Conclusion HS3ST3B1 can inhibit HBV replication and reduce the level of HBV total RNA,but the downregulation of HBV total RNA may not be the result of direct intereaction of HS3ST3B1 and HBV promoters.