1，4-苯醌暴露下小鼠骨髓细胞p15基因表达和甲基化状态

mRNA expression and methylation status of p15 promoter in mouse bone marrow cells exposed to 1,4- benzoquinone

目的探讨1，4-苯醌（1，4-BQ）暴露下，体外原代培养的小鼠骨髓细胞中抑癌基因p15的mRNA表达及启动子区的甲基化状态。方法体外分离小鼠骨髓细胞，测定0、0．1、1．0、5．0、10．0、20，0、40．0μmol／L的1，4-BQ对小鼠骨髓细胞活力的影响，最终确定以0、0．1、1．0、10．0μmol／L的1，4-BQ染毒骨髓细胞24h；实时荧光定量聚合酶链反应（PCR）检测p15基因mRNA的表达变化；重亚硫酸盐测序法（BSP）检测启动子区域CpG岛的甲基化状态。结果1，4-BQ对小鼠骨髓细胞具有剂量依赖的毒性作用，其LC50为8．3μmol／L（95％CI：4．6～10．6μmol/L）。与对照组（100．O％）比较，10．0、20．0、40．0p,mol／L1，4-BQ染毒组小鼠的骨髓细胞生仔率（35．9％、35．8％、30．5％）均明显降低，差异有统计学意义（P〈0．01）。10．0μmol／L染毒组P15mRNA表达为对照组的43％，与对照组比较，1．0和10．0p,mol／L染毒组p15基因mRNA表达量明显下降，差异有统计学意义（P〈0．05或P〈0．01）：BSP测序结果显示，染毒组和对照组相同，p15基因启动子区域CpG岛上56个甲基化位点仍保持非甲基化状态。结论1，4-BQ暴露可导致原代培养的小鼠骨髓细胞中p15基因的mRNA表达量下降，但启动子区域CpG岛的甲基化状态未受影响，提示甲基化不参与1，4-BQ介导的p15基因表达下调.

Objective To detect the expression and the CpG island methylation status of tumor sup- pressor gene p15 after exposure to 1,4-benzoqninone （1,4-BQ） in primary cultivated C57BL/6J mouse bone marrow cells in vitro. Methods The mouse bone marrow cells were isolated in vitro. The effect of 0, 0.1, 1, 5, 10, 20, and 40 μmol/L 1,4-BQ on cell viability （CKK-8） was detected. 0, 0.1, 1, 10 μmol/L 1,4-BQ were used to intoxicate the mouse bone marrow cells for 24 h, Real-time PCR was employed to analyze the mRNA expression level of p 15; The bisulfite sequencing PCR （BSP） was used to look into the methylation status of CpG islands in p15 promoter region. Results 1,4-BQ exhibited dose-dependent toxicity to mouse bone marrow cells, and the LC50 was 8.3 μmol/L （95% Ch 4.6-10.6 μmol/L）. The mRNA expression of p15 in 10 μmol/L group was only equivalent to 43% of control group. Compared with control group, the decrease of p15 mRNA expression in 1 and 10 μmol/L concentration were obvious, and the differences had statistical significance （P〈 0.05 or P〈0.01 ）. BSP sequencing results were same between the exposure groups and control group, the 56 CpG sites on CpG islands remained in the state of unmethylated. Conclusion mRNA expression of p15 gene decreases after exposure to 1,4-BQ, but the CpG islands menthylation status in promoter is not affected, suggesting that methylation does not participate in 1,4-BQ-mediated p15 gene expression decrease, other effect mechanisms still need to be,investigated.