文档介绍：三氧化二砷对MRL/lpr狼疮小鼠脾脏CD4+T细胞IFN-γ表达及其mRNA
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作者:陈培荣,朱载华,邹正平,朱小春
【摘要】目的:观察三氧化二砷(ATO)对MRL/lpr狼疮小鼠脾脏CD4+T细胞分泌IFN-γ及其mRNA的影响。方法: 免疫磁珠分别分选18~20周MRL/lpr狼疮小鼠和C57BL/6J正常对照小鼠脾脏CD4+T细胞,采用流式细胞术鉴定细胞纯度。PHA-p(20μg/mL)和IL-2(1 000 IU/mL)常规刺激48 h后进行如下实验:(1)不同浓度ATO(、)作用24 h;(2)1μmol/LATO作用不同时间(12、24和48 h);(3)不同药物处理组:①PBS组:空白对照;②ATO组:1μmol/LATO;③5-氮杂胞苷(5-AzaC)组:1μmol/L 5-AzaC;④ATO+5-AzaC组:1μmol/LATO+1μmol/L 5-AzaC。酶联免疫吸附法(ELISA)测定培养上清液IFN-γ的表达量;实时荧光定量PCR法检测不同药物处理组中IFN-γmRNA的表达情况。结果:① h对细胞存活率无明显影响。②1 mmol/lATO作用24 h能抑制MRL/lpr小鼠脾脏CD4+T细胞IFN-γ的转录和翻译水平。③经5-AazC处理后MRL/lpr和C57BL/6J小鼠脾脏CD4+T细胞IFN-γ的转录和翻译水平升高,1 mmol/LATO作用24 h能抑制其异常活化状态。
④MRL/lpr小鼠脾脏CD4+T细胞IFN-γ的分泌和表达水平均较C57BL/6J小鼠明显升高。结论: 1 mmol/L ATO作用24 h能在一定程度上有效抑制活化状态下MRL/lpr小鼠脾脏CD4+T细胞IFN-g的异常分泌,下调其mRNA表达水平而不影响其存活率。
【关键词】系统性红斑狼疮;三氧化二砷;5-氮杂胞苷;γ-干扰素
Abstract: Objective: To investigate the effects of arsenic trioxide (ATO) on the levels of IFN-γexpression and its mRNA transcripts in splenic CD4+T cells from MRL/lpr mice. Methods: CD4+T cells were isolated from the spleens of MRL/lpr and C57BL/6J mice as control via ic bead separation at 18 to 20 weeks of age. Cell purity was checked by flow cytometry. CD4+ T cells were stimulated for 48 h in the presence of PHA-p(20μg/ml)and IL-2(1 000 IU/ml). Then cells were treated with ATO(, and ) for 24 h,which were also exposed to ATO treatment for 12,24 and 48 h. In addition,cells were added with ATO,or 5-azacytidine (5-AzaC) or bined with 5-AzaC at ,respectively,pared to those given PBS. Enzyme linked immunosorbent assay(ELISA)was used to measure serum levels of IFN-γ. A real-time fluorescent quantitative PCR (FQ-PCR) was set up to determine IFN-
γ mRNA expression in different drugs-treated-CD4+T cells. Results: ①ATO( μmol/L) was of no effect on survival rate of cells cultured for 24 h.②After treated with ATO for 24 h,transcription and interpretation levels of IFN-γin spl