As_2O_3/TRAIL诱导的骨髓增生异常综合征原始细胞系MDS-L的生物学变化

Biologic changes in MDS-L cell line induced by As_2O_3 and /or TRAIL

目的 观察骨髓增生异常综合征 (MDS)髓系原始细胞系MDS L经不同剂量和不同时间的三氧化二砷 (As2 O3 )和肿瘤坏死因子相关凋亡诱导配体 (TRAIL)处理后的生物学变化。方法 体外培养的MDS L细胞经 9种不同浓度的药物及药物组合 (As2 O3 :1,2 ,5mmol/L ;TRAIL :10 0 ,30 0 ,5 0 0 μg/L ,As2 O3 1mmol/L +TRAIL 10 0 μg/L ;As2 O3 2mmol/L +TRAIL 30 0 μg/L ;As2 O3 5mmol/L +TRAIL 5 0 0 μg/L)处理 ,在 2 4 ,4 8和 72h后收获细胞。对未经药物处理的细胞和药物处理后收获的细胞均进行流式细胞仪检测细胞凋亡 ;药物处理 2 4h后的细胞再经体外培养 18d后作形态学观察 ,同时以流式细胞仪检测CD34+ 细胞变化 ,检测药物的促分化作用 ;RT PCR检测P15 ink4b mRNA表达 ;甲基化特异性PCR(MethylationspecificPCR ,Msp)检测P15 ink4bDNA甲基化状态 ;DAB免疫酶标检测P15 ink4b蛋白水平表达。结果 不同的药物组合均可诱导细胞发生凋亡 ,药物处理 4 8h凋亡达高峰 (约 2 5 % ) ,72h时仍有约9%的细胞凋亡。药物处理 (尤其是As2 O3 +TRAIL)导致细胞明显的形态学分化 ,而TRAIL能显著降低CD34+ 细胞比率。未经处理的MDS L细胞基本不表达P15 ink4b,并伴有明显的DNA甲基化。药物处理后P15 ink4b表达增强 。

Objective To investigate the biological changes in myelodysplastic syndromes (MDS) myeloid blast cell line MDS-L after different duration and concentration of As_(2)O_(3 )/TRAIL (TNFa related apoptosis inducing ligand) treatment. Methods MDS-L cells were treated with As_(2)O_(3 ) and TRAIL at 9 different concentrations and the treated cells were detected at 24 h, 48 h and 72 h for biologic indexes. The same detections were conducted in untreatd MDS-L cells and normal and MDS marrow cells as controls. Apoptosis was assayed by flow cytometry after AnnexinV-FITC labelling. Differentiation-induction effect of these drugs on the cells were detected by morphologic examination and CD34^+ proportion analysis after 24 hours treatment and further agar culture for 18 days; P15^(ink4b) mRNA expression were detected by RT-PCR and its protein expression by DAB immunocytochemistry, P15^(ink4b) DNA methylation by methylation specific PCR (Msp). Results As_2O_3/TRATL treatment promoted MDS-L cells to undergo apoptosis and As_2O_3 plus TRATL showed obvious differentiation-induction effect on MDS-L. P15^(ink4b) mRNA expression was upregulated in MDS-L cell line after different drug treatment but almost no protein expression increased. Increased P15 expression seemed to be related with DNA demethylation effect of these drugs. Conclusions As_2O_3 or /and TRAIL treatment could promote apoptosis of the clonal cells and induce incomplete cell differentiation. The drugs treatment could also increase P15^(ink4b) expression in MDS-L cell line through their demethylation effects. [