硫酸类肝素-3-0-磺基转移酶B1对乙型肝炎病毒复制的影响

Down-regulation of hepatitis B virus replication by heparin sulfate-D-glucosaminyl-3-O-sulfotrans3B1ferase

目的研究硫酸类肝素3O磺基转移酶Bl（HS3ST381）对HBV复制的影响。方法以HepG2细胞为阴性对照组，转染2．5ugpCH9HBV（HBV表达质粒）的HepG2细胞为阳性对照组；转染了2．5μgpCH9HBV、1．5μgpcDNA3．1（HS3ST3Bl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2．5μgpCH9HBV、1．5μgpCDNA3．1-HS3ST381和2．0μgpTZU6＋1的HepG2细胞为实验组A；转染了2．5μgDCH9-HBV、1．5ugpCDNA3．1-HS3ST3Bl和2．0μgpshll26（HS3ST3Bl的干扰质粒）的HepG2细胞为干扰组A。转染了2．5μgpCH9-HBV和2．0μgpTzu6＋1的HepG2细胞为实验组B；转染了2．5μgpCH9HBV和2．0μgpshll26的HepG2为干扰组B。采用Southernblot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l（spl）、包膜蛋白启动子2（sp2）]活性的关系。采用SPSSl7．0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HBVDNA含量为l，实验组A、干扰组AHBVDNA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BHBVDNA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、1．5ug时，HBVDNA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，尸值分别为0．029、0．041、0．015，差异均有统计学意义。实验组AHBV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AHBV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。

Objective 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＋I; （4） Treatment A, transfected with pcHg-HBV ＋ pCDNA3.1-HS3ST3B 1 ＋ pTZU6＋ 1; （5） Interference A, transfected with pCH9-HBV ＋ pCDNA3.1-HS3ST3B 1 ＋ psh1126 （a plasmid to interfere HS3ST3B 1 expression）; （6） Treatment B, transfected with pCH9-HBV ＋ pTZU6＋ 1; （7） Interference B, transfected with pCH9-HBV ＋ pshl 126. 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 promoterl（spl）, surface antigen promoter2 （sp2）] were assayed by Dual-Luciferase Reporter Assay System. The data was analyzed using one way ANOVA, with P 〈 0.05 indi- cating 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 ceils were transfected with 0.5, 1.0, 1.5 μg 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 I--IBV promoters activity. Conclusion HS3ST3B1 can inhibit I-IBV 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 HS3ST3B 1 and HBV promoters.