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(1000IU/mL)常规刺激48h后进行如下实验:(1)不同浓度ATO(0.5、1.0和2.0μmol/L)作用24h;(2)1μmol/LATO作用不同时间(12、24和48h);(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/L5-AzaC;④ATO+5-AzaC组:1μmol/LATO+1μmol/L5-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:①1.0μmol/LATO作用24h对细胞存活率无明显影响。②1mmol/lATO作用24h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,1mmol/LATO作用24h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:1mmol/LATO作用24h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。
Objective： To investigate the effects of arsenic trioxide （ATO） on the levels of IFN-γ expression and its mRNA transcripts in splenic CD4＋T cells from MRL/lpr mice. Methods： CD4＋T cells were isolated from the spleens of MRL/1pr and C57BL/6J mice as control via magnetic 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（1000IU/ml）. Then cells were treated with ATO （0.5, 1.0 and 2.0μmol/L） for 24 h, which were also exposed to 1.0μmol/L ATO treatment for 12, 24 and 48 h. In addition, cells were added with ATO, or 5-azacytidine （5-AzaC） or ATO combined with 5-AzaC at 1.0μmol/L, respectively, as compared to those given PBS. Enzyme linked immunosorbent assay （ELISA） was used to measure serum levels of IEN-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（1.0μmol/L） was of no effect on survival rate of cells cultured for 24 h.②After treated with 1.0μmol/L ATO for 24 h, transcription and interpretation levels of IFN-γ in splenic CD4＋T ceils from MRL/lpr mice were inhibited.③In the presence of 5-AazC, the levels mentioned above markedly increased in MRL/1pr and C57BL/6J mice, which could be also inhibited by 1.0μmol/L ATO after a 24-h incubation.④The production and mRNA expression of IFN-γ of splenic CD4＋T cells were significantly higher in MRL/1pr mice,compared to C57BL/6J mice. Conclusion： The treatment of splenic CD4＋T ceils with 1.0 μmol/L ATO by culturing for 24 h could efficiently inhibit abnormal secretion level of IFN-γ in MRL/1pr mice, and down-regulate mRNA level without affecting the survival rate.
出处
《温州医学院学报》
CAS
2009年第4期333-337,共5页
Journal of Wenzhou Medical College
