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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 ,并伴有DNA去甲基
Objective To investigate the biological changes of MDS myeloid primary cell line MDS L treated with different doses of As 2 O 3 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Methods MDS L cells cultured in vitro were treated with 9 different concentrations of drugs and drugs (As 2 O 3: 1, 2, 5 mmol / L, TRAIL: 100, 300, 500 μg / L, As 2 O 3 1 mmol / L + TRAIL 10 0 μg / L; As 2 O 3 2 mmol / L + TRAIL 30 0 μg / L; As 2 O 3 5 mmol / L + TRAIL 500 μg / L) and cells were harvested at 24, 48 and 72 hours. Cell apoptosis was measured by flow cytometry after drug-treated cells and drug-harvested cells were treated; drug-treated cells were cultured for 24 hours and then cultured for 18 days. Morphological changes were observed by flow cytometry CD34 + cells were detected by flow cytometry. The chemotaxis of P15 and ink4b was detected by RT-PCR. The methylation status of P15 and ink4b was detected by Methylationspecific PCR (MSP). The protein expression of P15 and ink4b was detected by DAB immunoenzyme. Results Different combinations of drugs could induce cell apoptosis, the peak of apoptosis was reached 48 h after drug treatment (about 25%), and there was still about 9% apoptosis at 72 h. Drug treatment (especially As2 O3 + TRAIL) resulted in significant morphological differentiation of cells, whereas TRAIL significantly reduced the percentage of CD34 + cells. Untreated MDS L cells did not express P15 ink4b at all, with significant DNA methylation. P15 ink4b expression increased after drug treatment, accompanied by DNA to methyl