摘要
目的:观察三氧化二砷(ATO)对MRL/lpr狼疮小鼠脾脏CD4+T细胞IFN-γ基因启动子甲基化的影响。方法:免疫磁珠法分选16~18周MRL/lpr狼疮小鼠和C57BL/6J正常对照小鼠脾脏CD4+T细胞,PHA-p(20μg/mL)和IL-2(1 000 IU/mL)刺激48 h后分为以下不同药物处理组:①PBS组:空白对照;②ATO组:1μmol/L ATO作用24 h;③ATO+5-氮杂胞苷(5-AzaC)组:1μmol/L 5-AzaC作用72 h后1μmol/L ATO作用24 h。CD4+T细胞经过以上处理后抽提基因组DNA,亚硫酸氢盐修饰,PCR扩增IFN-γ基因启动子,产物凝胶回收,克隆入pMD-19T载体,转化入E.coli DH5α,筛选阳性克隆测序,测序结果采用生物信息学软件进行分析。结果:①MRL/lpr小鼠脾脏CD4+T细胞IFN-γ基因启动子呈低甲基化水平。②1μmol/LATO作用24 h后MRL/lpr小鼠脾脏CD4+T细胞IFN-γ基因启动子甲基化水平明显增高。③1μmol/L ATO作用24 h能使经过5-AzaC干预后的MRL/lpr小鼠脾脏CD4+T细胞IFN-γ基因启动子甲基化水平明显增高。结论:1μmol/L ATO作用24 h能在一定程度上有效逆转活化状态下MRL/lpr小鼠脾脏CD4+T细胞IFN-γ基因启动子低甲基化。
Objective:To observe the effects of arsenic trioxide(ATO) on the methylation status of the IFN-γ gene promoter of splenic CD4+T cells from MRL/lpr mice.Methods:CD4+T cells were isolated from the spleens of MRL/lpr and C57BL/6J mice at the age of 16 to 18 weeks via magnetic activated cell sorting(MACS),and it’s cell purity was checked by fluorescence activated cell sorting(FACS).CD4+T cells were stimulated in vitro for 48 h in the presence of PHA-p(20μg/mL) and IL-2(1 000 IU/mL).The cultured CD4+T cells were divided randomly into 3 groups:control group(treated with PBS),ATO group(treated with 1μmol/L ATO for 24 h) and ATO+5-azacytidine(5-AzaC) group(treated with 1μmol/L 5-AzaC for 72 h before 1μmol/L ATO for 24 h).After treated as described above,the genomic DNA was isolated,bisulfite-treated.The IFN-γ gene promoter was amplified in nested PCR.PCR products were separated on agarose gels,excised,cloned,sequenced and analyzed by bioinformatics software on line.Results:①IFN-γ gene promoter of splenic CD4+T cells from MRL/ lpr mice was hypomethylated.②Methylation of IFN-γ gene promoter of splenic CD4+T cells from MRL/ lpr mice was enhanced after treated with ATO.③Methylation of IFN-γ gene promoter of splenic CD4+T cells prestimulated with 5-AzaC was also enhanced after treated with ATO.Conclusion:The treatment of splenic CD4+T cells of MRL/lpr mice with 1μmol/L ATO in vitro by culturing for 24 h can efficiently improve the methylation status of the IFN-γ gene promoter.
出处
《温州医学院学报》
CAS
2010年第3期231-234,238,共5页
Journal of Wenzhou Medical College
基金
温州市科技计划基金资助项目(Y20090240)