氟达拉滨诱导多发性骨髓瘤细胞凋亡及其基因表达分析

目的 探讨氟达拉滨诱导多发性骨髓瘤(MM)细胞凋亡及其分子机制.方法 用MTT法检测氟达拉滨对MM细胞的生长抑制作用;用流式细胞术检测细胞凋亡;Western blot法分析凋亡蛋白表达;采用细胞凋亡基因芯片技术检测并分析对照组与氟达拉滨处理组间的基因表达差异.结果 氟达拉滨显著抑制RPMI8226和KM3细胞生长,呈时间和剂量依赖性.24 h半数生长抑制值(IC50)分别为2.13μg/ml和0.36 μg/ml.流式细胞术分析显示,氟达拉滨作用24 h后MM细胞凋亡呈剂量依赖性,同时伴有caspase-3和PRAP激活,具有典型的细胞凋亡生物学特征.在97个凋亡相关基因中,筛选出25个差异表达基因.氟达拉滨处理组与对照组比较,表达上调的基因有13个,主要涉及Bcl-2家族促凋亡基因、肿瘤坏死因子及其受体超家族基因,以及胱天蛋白酶募集结构域家族.表达下调的基因有12个,主要涉及Bcl-2家族抗凋亡基因、肿瘤坏死因子超家族及其受体相关因子基因,以及凋亡抑制蛋白家族.结论 氟达拉滨显著抑制MM细胞生长,诱导细胞凋亡;其作用机制涉及多个凋亡相关基因表达改变.

Effects of fludarabine on apoptosis and gene expression profile in multiple myeloma cells

Objective To investigate the in vitro effects of fludarabine on apoptosis and gene expression profile in human multiple myeloma(MM)cells.Methods Cell growth was measured by a MTT assay.Cells apoptosis was evaluated by flow cytometry.The activation of caspase cascade was determined by Western blot analysis.The expression profile of apoptosis-related genes in human MM cells was detected by cDNA expression array system.Results The growth of RPMI8226 and KM3 cells was suppressed by fludarabine treatment in a dose and time-dependent manner.After treatment with fludarabine for 24 h,the IC50 for RPMI8226 cells was 2.13 μg/ml,and 0.36 μg/ml for KM3 cells.Apoptotic cells of RPMI8226 and KM3 increased in a dose-dependent manner after exposure to fludarabine for 24h.Western blot analysis showed the activation of caspase-3 and PARP in the MM cells treated with fludarabine.The cDNA expression array showed that multiple pathways were involved in the apoptosis induced by fludarabine.Among 97 apoptosis-related genes,25 genes were differently expressed and 13 expressions were up-regulated in fludarabine treated group,involving in Bcl-2 pro-apoptotic gene,tumor necrosis factor(TNF)and its receptor superfamily gene,and caspase recruitment domain family,12 genes expression down-regulated,including Bcl-2 antiapoptotic gene,TNF superfamily and its receptor related factor gene and apoptosis inhibitor protein family.Conclusion Fludarabine can significantly inhibit MM cell growth and induce apoptosis in vitro.The multiple pathways may be involved for the apoptosis in MM cells.