目的分析镉染毒处理肝细胞中蛋白磷酸酶2A(PP2A)-Aα支架亚基基因PPP2r1A启动子区甲基化状态及其转录水平的改变。方法采用永生化人正常肝l02细胞及肝细胞癌HEP g2细胞为研究对象,对其进行以下分组和处理:1低、中和高剂量氯化镉(Cd Cl2)处理组,分别予浓度为20.0、40.0和60.0μMOl/l Cd Cl2处理24 H;2低、中和高剂量5-氮杂-2’-脱氧胞苷(5-AzA-d C)处理组,分别予浓度为2.5、5.0和10.0μMOl/l 5-AzA-d C处理48 H;3 5-AzA-d C组予浓度为5.0μMOl/l的5-AzA-d C处理48 H,Cd Cl2组予浓度为40.0μMOl/l的Cd Cl2处理24 H,(5-AzA-d C+Cd Cl2)组予浓度为5.0μMOl/l的5-AzA-d C预处理48 H后再予浓度为40.0μMOl/l Cd Cl2处理24 H;4 Cd Cl2处理组予浓度为40.0μMOl/l Cd Cl2处理24 H。上述4种分组均设对照组,予等体积生理氯化钠溶液或二甲基亚砜处理。经1~3处理后的细胞采用实时荧光定量聚合酶链反应(PCr)检测PPP2r1A、金属硫蛋白1b(MT1b)和dnA甲基转移酶3A(dnMT3A)的MrnA转录水平(以对照组水平为1.00)。经4处理后的细胞采用亚硫酸氢盐修饰后PCr扩增PPP2r1A启动子区克隆测序检测CP g岛的甲基化情况。结果 l02细胞和HEP g2细胞中,不同剂量Cd Cl2处理组PPP2r1A MrnA转录水平随镉处理剂量增高呈剂量依赖性下降(P0.05)。结论外源化学物Cd Cl2可诱导肝细胞中PPP2r1A转录水平降低,可能与镉能够引起目的基因启动子区甲基化状态改变有关,提示PP2A亚基基因的表观遗传学调控可影响镉诱导的肝细胞功能。Objective To analyze the effects of cadmium on the promoter methylation and transcriptional level of protein phosphatase 2A( PP2A)-Aα supported subunit gene PPP2R1 A gene in hepatocytes.Methods The immortalized human fetal liver cell line L02 and the hepatocellular carcinoma cell line Hep G2 were selected as the research objects: 1 Cells were treated with low-,medium- and high-dose( 20.0,40.0 and 60.0 μmol / L) cadmium chlorid( Cd Cl2) for 24 h.2Cells were treated with low-,medium- and high-dose( 2.5,5.0 and 10.0 μmol / L) 5-aza-2'-deoxycytidine( 5-Aza-d C)for 48 h.3 Cells were given 5.0 μmol / L for 48 h in 5-Aza-d C group,cells were exposed to 40.0 μmol / L Cd Cl2 for 24 h in Cd Cl2 group and cells were exposed to 40.0 μmol / L Cd Cl2 for 24 h after 48 h pretreatment of 5.0 μmol / L 5-Aza-d C in( 5-Aza-d C + Cd Cl2) group.4 Cells were treated with 40.0 μmol/L Cd Cl2 for 24 h in Cd Cl2 group.The above groups were all given the controls with the same volumes of physiological sodium chloride solution or dimethyl sulfoxide.Real-time fluorescent quantitative polymerase chain reaction( PCR) detection was used to detect the mRNA transcriptional levels of PPP2R1 A,Metallothionein 1B( MT1B),DNA methyltransferase 3A( DNMT3A) after treatments 1-3.After treatment4,cloning sequencing was used to detect the Cp G island methylation status of PPP2R1 A promoter after bisulfite sequencing PCR.Results In L02 and Hep G2 cells,the transcriptional levels of PPP2R1 A mRNA in Cd Cl2 group were decreased in a dose-dependent manner( P 0.05).Conclusion It was indicated the Cd Cl2 could lead to the transcription inhibition of PPP2R1 A,and the effect may be related with the change of its promoter methylation status.These data showed epigenetic regulation of PP2 A subunit genes may affect the function of hepatocytes exposed to cadmium.国家自然科学基金(81172705;81072334;81130052); 广东省自然科学基金重点项目(S2011020002769
