氧自由基诱导鸭乙型肝炎病毒DNA整合及其细胞传代研究

目的探讨氧自由基对鸭乙型肝炎病毒（DHBV）DNA在受感染细胞DNA上整合发生的影响以及整合后DHBVDNA的细胞传代性。方法选择鸡肝癌细胞株—LMHD21-6，在其由单个细胞培养至3×107～5×107个细胞（共23代）的过程中，用低浓度H2O2（10.0μmol/L）作用于细胞；TUNEL法检测细胞DNA链断裂情况；Southernblot技术观察细胞DNA上新的整合型DHBVDNA的发生情况。结果(1)低浓度H2O2（≤10.0μmol/L）导致的细胞死亡率仅为32.0%，但可诱导细胞DNA链断裂致细胞调亡，而H2O2浓度＞10.0μmol/L时其细胞死亡率＞50.0%；(2)低浓度H2O2作用的细胞DNA上新的DHBVDNA整合发生率为50.0%（6/12），而无H2O2作用的发生率仅8.33%（1/12），两者比较差异有显著意义（P＜0.05＝；(3)含新的整合型DHBADNA的细胞在无H2O2作用下，再由单个细胞培养至第23代，其子代细胞DNA上可见与其母代细胞DNA上新的整合型DHBVDNA碱基大小完全一致的DHBVDNA带。结论氧自由基为DHBVDNA整合发生的重要诱因之一，新的整合型DHBVDNA可有较稳定的细胞传代。

Duck hepatitis B virus DNA integration induced by oxidative radicals and its transmission from parent cells to offspring ones

OBJECTIVE: To investigate the role of oxidative radicals in duck hepatitis B virus DNA (DHBV DNA) integration and the transmission of integrated DHBV DNA from parent cells to their offspring cells. METHODS: TUNEL assay and Southern blot hybridization were used to detect the cellular DNA strand breaks and the incidence of new type of DHBV DNA integration into cellular DNA of LMHD(21-6) cell line after the cells treated by low dose of hydrogen peroxide (10.0 micromol/L) during its growth from one cell to 3 +/- 10(7) to 5 +/- 10(7) cells. RESULTS: The rate of cell death induced by low dose of H(2)O(2) (10.0 micromol/L) was 32.0%, but that induced by high dose (>10.0 micromol/L) was more than 50.0%. At the same time, the incidence of new DHBV DNA integration into cellular DNA induced by 10.0 micromol/L of H(2)O(2) was 50.0% (6/12) and that in control group was 8.33% (1/12). There was a significant difference between the two groups (P<0.05). Besides, the new integrated DHBV-DNA in the parent cells could be found in the DNA of their offspring cells with the same size of base pair. CONCLUSION: Oxidative radicals is one of the important inducing factors in DHBV-DNA integration into cellular DNA and the integrated DHBV-DNA is able to transmit from the parent cells to their offspring cells stably.