目的:探讨慢性粒细胞白血病(CML)急变中hPer3(human period 3)基因启动子甲基化检测的临床意义以及诱导hPer3表达对CML细胞株K562的作用.方法:应用甲基化特异性PCR(MS-PCR)和实时荧光定量PCR检测29例CML患者骨髓hPer3基因启动子甲基化状态和mRNA表达,以40例缺铁性贫血(IDA)患者骨髓作为对照.将pcDNA3.1-hPer3表达质粒经脂质体介导转染CML细胞株K562,MTT法检测生长抑制率,Annexin V/PI染色法检测细胞凋亡.结果:IDA组、CML慢性期、CML加速期和CML急变期中,hPer3启动子甲基化阳性率(例)分别为0%(0/40)、17.65%(3/17)、66.67%(4/6)和83.33%(5/6),CML急变期和加速期甲基化阳性率均高于CML慢性期,差异显著(2=8.44,P＜0.01;2=5.03,P＜0.05).对照组和CML慢性期、CML加速期和CML急变期中,hPer3mRNA表达分别为75.03±18.16和5.13±2.29、0.40±0.18、0.17±0.05,两两之间差异显著(P＜0.01).与空载体转染组相比,hPer3转染后各时点的细胞抑制率和凋亡率均升高(P＜0.01).结论:CML中hPer3启动子高甲基化导致了基因表达沉默,可能作为CML急变的监测指标.hPer3的过表达能抑制CML细胞株增殖,并促进其凋亡.%AIM; To investigate the clinical significance of the methylation status of human period 3 (hPer3) promoter in the patients with chronic myelocytic leukemia (CML). The effect of induction of hPer3 expression on K562 cells was also observed. METHODS: The methylation status of hPer3 promoter and Mrna expression in bone marrow of 29 CML patients and 40 iron - deficiency anemia (IDA ) patients were detected by methylation - specific PCR (MS - PCR) and real - time PCR. A plasmid containing hPer3 Cdna and an empty plasmid were transfected into K562 cells by cationic liposomes. The inhibitory rates of cell proliferation were determined by MTT assay, and the apoptotic rates were measured by Annexin V/PI staining. RESULTS: The positive rates of hPer3 promoter methylation in IDA group, CML chronic phase group, CML accelerated phase group and CML blastic phase group were 0% (0/40) , 17.65% (3/17), 66.67% (4/6) and 83.33% (5/6), respectively. The positive rates hPer3 promoter methylation in CML blastic phase group and CML accelerated phase group were significantly higher than that in CML chronic phase group (X2=8.44, P <0.01; X2 =5.03, P <0. 05). The positive rates of hPer3 promoter methylation in CML blastic! Phase group, CML accelerated phase group and CML chronic phase group were significantly higher than that in IDA group (X2= 29.29, P < 0. 01; X2 = 21.41, P < 0.01; X2 = 4.33, P < 0.05 ). The Mrna expression levels in control group, CML chronic phase group, CML accelerated phase group and CML blastic phase group were 75.03 ± 18.16, 6. 84 ±2.54, 0.44 ±0.21 and 0. 13 ±0.09, respectively, and significant differences between any 2 group were observed (P < 0.01 ). Compared with empty plasmid transfection group, the inhibitory rates and the apoptotic rates were significantly higher at each time point after transfection ( P < 0. 01). CONCLUSION: Hypermethylation of hPer3 promoter, which leads to gene silencing, may be an indicator of CML monitoring. In K562 cells, overexpression of hPer3 inhibits the cell growth and induces cell apoptosis.
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