磷酸脂酶抑制剂冈田酸对HepG2.2.15细胞中乙型肝炎病毒复制的影响

 目的 初步鉴定可能与 HBV 核心蛋白去磷酸化有关的磷酸酯酶。 方法 以磷酸酯酶 2A 抑制剂冈田酸（OA）处理体外培养的人肝癌 HepG2.2.15 细胞。将细胞分为 4 组，分别加入终浓度为 0（对照组）、10、20、30 ng/ml 的 OA（OA 10 ng 组、OA 20 ng 组、OA 30 ng 组），每组各设 5 孔。在培养 0、2、4、6 d 时分别收集各组细胞培养上清液，采用电化学发光方法检测 HBsAg 浓度，荧光实时定量 PCR 方法检测 HBV DNA 拷贝数。取培养 2 d 时的各组细胞，采用蛋白质印迹方法检测 HBcAg 异质性，免疫荧光细胞化学染色方法检测 HBcAg 亚细胞定位。 结果 OA 10ng 组、OA 20 ng 组各时间点细胞培养上清液HBsAg 浓度、HBV DNA 拷贝数分别与对照组相应各时间点比较，差异均无统计学意义；在培养 2 d 时 HBcAg 电泳条带、亚细胞定位分别与对照组比较均无明显异常。OA 30 ng 组在培养 2、4、6 d 时细胞培养上清液 HBsAg 浓度（ng/ml）均明显低于对照组相应各时间点（分别为 385.9 ± 43.1 vs. 510.2 ± 63.3、346.5 ± 39.6 vs. 484.6 ± 59.4、295.1 ± 32.8 vs. 467.2 ± 52.9，P 值分别为 0.028、0.022、0.016）；在培养 2 d 时细胞培养上清液 HBV DNA 拷贝数（ml-1）明显高于对照组相应时间点（6.71 ± 6.07 vs. 6.34 ± 5.72，P = 0.022），而在培养 4、6 d 时均明显低于对照组相应各时间点（分别为 5.80 ± 5.19 vs. 6.29 ± 5.68、4.75 ± 4.15 vs. 6.25 ± 5.58，P 值分别为 0.008 和 0.005）；在培养 2 d 时 HBcAg 电泳条带明显增宽，约 20% 的 HepG2.2.15 细胞的细胞核内 HBcAg 红色荧光信号明显增强。 结论 短时间、高浓度的 OA 处理可提高 HBV DNA 的复制水平和核心蛋白的磷酸化水平，提示磷酸酯酶 2A 可能参与了 HBV 核心蛋白的去磷酸化过程。

Effect of phospholipase inhibitor okadaic acid on HBV replication in HepG2.2.15 cells

Objective To identify the phospholipase involved in the dephosphorylation of core protein in HBV life cycle. Methods Human hepatocarcinoma cell line HepG2.2.15 was divided into 4 groups, and respectively treated with 0, 10, 20, or 30 ng/ml okadaic acid (OA) as the control, OA 10 ng, OA 20 ng, and OA 30 ng groups (5 wells in each group). Then the cells culture supernate was collected on days 0, 2, 4, and 6. The concentrations of HBsAg and HBV DNA in the cells culture supernate were determined by electrochemiluminescence and real-time fluorescence PCR, respectively. The heterogeneity and subcellular distribution of HBcAg in the cells collected on day 2 was detected by fluorescence immunocytochemistry and western blot. Results At any time point, no significant difference was observed between the OA 10 ng and OA 20 ng groups and the control in the concentrations of HBsAg and HBV DNA, as well as in the heterogeneity and subcellular distribution of HBcAg on day 2. Whereas, in the OA 30 ng group, the concentration of HBsAg (ng/ml) was significantly lower than that in the control on days 2, 4 and 6 [(385.9 ± 43.1) vs. (510.2 ± 63.3), P = 0.028; (346.5 ± 39.6) vs. (484.6 ± 59.4), P = 0.022; and (295.1 ± 32.8) vs. (467.2 ± 52.9), P = 0.016]. On day 2, the concentration of HBV DNA (ml-1) in the OA 30 ng group was significantly higher than that in the control [(6.71 ± 6.07) vs. (6.34 ± 5.72), P = 0.022], while on days 4 and 6, it was significantly lower [(5.80 ± 5.19) vs. (6.29 ± 5.68) and (4.75 ± 4.15) vs. (6.25 ± 5.58), P = 0.008 and 0.005, respectively). In the OA 30 ng group, band broadening in electrophoresis was observed on day 2, meanwhile the intensity of HBcAg red signals in the nuclei was markedly increased in approximately 20% of the HepG2.2.15 cells. Conclusions Short-term and high-concentration OA treatment can increase the levels of HBV DNA replication and core protein phosphorylation in HepG2.2.15 cells. Phospholipase 2A may play a role in core protein dephosphorylation during HBV life cycle.