三氧化二砷对MRL/lpr狼疮鼠IFN-γ基因表达的调控

Effects of arsenic trioxide on IFN-γ gene expression in MRL/lpr mice

目的:探讨三氧化二砷(ATO)调控MRL/lpr狼疮鼠干扰素γ(IFN-γ)基因表达的机制。方法:将20周龄MRL/lpr狼疮鼠和正常C57BL/6J小鼠无菌条件下取出脾脏,制成脾脏淋巴细胞悬液。体外经植物血球凝集素P(PHA-P,终浓度20 mg/L)和白细胞介素-2(IL-2,终浓度106IU/L)常规刺激48 h后,随机分为PBS组(空白对照)和ATO(1.0μmol/L)组,继续培养24 h。酶联免疫吸附法(ELISA)测定各组培养上清液IFN-γ的表达量,采用实时荧光定量PCR(Q-PCR)检测各组IFN-γmRNA的表达情况,应用基于半定量PCR和Q-PCR的染色质免疫共沉淀(ChIP)技术检测各组细胞IFN-γ启动子区乙酰化组蛋白H3、H4(acH3,acH4)及启动子区结合RNA聚合酶Ⅱ(RNAPⅡ)的水平。结果:(1)MRL/lpr狼疮鼠PBS组IFN-γ的分泌和IFN-γmRNA的表达均高于C57BL/6J小鼠PBS组(分别P<0.01和P<0.05),并且IFN-γ启动子区acH3、acH4的水平及启动子区域富集RNAPⅡ水平高于C57BL/6J小鼠PBS组(均P<0.01)。(2)在MRL/lpr狼疮鼠中,与PBS组相比,ATO组IFN-γ的分泌和IFN-γmRNA的表达下降(分别P<0.01,P<0.05),IFN-γ基因启动子区域acH3、acH4的水平及启动子区域富集RNAPⅡ水平也下降(均P<0.01)。(3)在C57BL/6J小鼠中,PBS组和ATO组之间以上指标均无差异。结论:ATO下调MRL/lpr狼疮鼠IFN-γ的分泌和IFN-γmRNA的表达可能是通过降低基因启动子区域acH3、acH4的水平减弱了RNAPⅡ依赖的转录,而ATO对正常C57BL/6J小鼠无明显影响。

AIM： To investigate the effect of arsenic trioxide（ATO） on interferon-γ（IFN-γ） gene expression in MRL/lpr mice.METHODS： The spleens were isolated from MRL/lpr mice and C57BL/6J mice at the age of 20 weeks under pathogen-free condition,and the cell suspensions were prepared.The splenic lymphocytes were stimulated in vitro for 48 h in the presence of phytohemagglutinin-P（PHA-P,20 mg/L） and IL-2（106 IU/L）,then were divided randomly into 2 groups： PBS group（treated with PBS for 24 h） and ATO group（treated with 1.0 μmol/L ATO for 24 h）.The concentration of IFN-γ in the supernatants of the cell culture was measured by ELISA.The mRNA level of IFN-γ was detected by real-time quantitative PCR（Q-PCR）.The chromatin immunoprecipitation（ChIP） combined with semiquantitative PCR and Q-PCR was used to detect the levels of acetylated histones H3 and H4（acH3 and acH4） at IFN-γ gene promoter region and the binding of RNA polymerase II（RNAPII） to IFN-γ promoter.RESULTS： Compared with PBS group of C57BL/6J mice,the secretion level of IFN-γ and the mRNA expression of IFN-γ were higher in PBS group of MRL/lpr mice（P0.01,P0.05 respectively）.The levels of acH3 and acH4 at IFN-γ gene promoter region and the binding of RNAPII to IFN-γ promoter were also higher in PBS group of MRL/lpr mice（P0.01）.In MRL/lpr mice,compared with PBS group,the secretion level of IFN-γ and the mRNA expression of IFN-γ were lower in ATO group（P0.01 and P0.05,respectively）.The levels of acH3 and acH4 at IFN-γ gene promoter region and the binding of RNAPII to IFN-γ promoter were also lower in ATO group（P0.01）.No difference of these indexes mentioned above between ATO group and PBS group in C57BL/6J mice was observed.CONCLUSION： ATO may down-regulate the secretion and the mRNA expression of IFN-γ by decreasing the level of acetylated histones at IFN-γ gene promoter region,which may have a negative effect on RNAPII-dependent transcription,and ATO has no effect on the normal mice.