早幼粒细胞白血病蛋白在髓系白血病细胞中的表达及对细胞增殖的影响

The expression of PML in chronic myeloid leukemia and effect on cell proliferation

目的探讨早幼粒细胞白血病（ promyelocyte leukemia, PML）蛋白在髓系白血病中的表达及其对人慢性粒细胞白血病细胞系K562细胞增殖的影响。方法采用实时荧光定量PCR技术检测了30例慢性粒细胞白血病（chronic myeloid leukemia, CML）患者及24名健康对照者外周血单个核细胞中PMLmRNA水平的差异。构建PML的真核表达载体，转染K562细胞后筛选出稳定表达PML的细胞株，以稳定表达空载体的细胞为对照。四甲基偶氮唑盐（MTT）法和流式细胞术分别检测PML过表达对细胞增殖和细胞周期的影响，并进一步用反转录-PCR（RT—PCR）和蛋白质印迹技术（Westernblot）检测增殖相关因子c-myc和p27kip1（p27）mRNA水平以及蛋白表达水平的改变。最后检测了CML患者及健康对照者外周血单个核细胞中c—myc和p27mRNA水平的差异。结果CML患者外周血单个核细胞中PMLmRNA表达水平（△Ct=9．02±0．74）明显低于健康对照组（△Ct=7．91±0．34），差异有统计学意义（t=5．07，P〈0．001）。PML过表达能明显抑制K562细胞增殖，引起细胞周期GO／G1期阻滞。PML稳定表达的K562细胞中，c—myc的mRNA和蛋白表达水平降低，p27的mRNA和蛋白表达水平升高。CML患者外周血单个核细胞中c—myc的mRNA表达水平（△Ct=7．13±0．43）显著高于健康对照组（△Ct=9．35±0．82），差异有统计学意义（t=6．78，P〈0．001），而p27的mRNA表达水平（△Ct=4．56±0．58）则显著低于健康对照组（△Ct=3．29±0．92），差异有统计学意义（t=2．93，P〈0．01）。结论PML在CML患者中低表达，PML可能通过调节c—myc和p27的表达从而抑制白血病细胞的增殖。

Objective To investigate whether PML is expressed differently in chronic myeloid leukemia （CML） patients and healthy controls, then explore the effect of PML on proliferation in leukemia cell lines K562. Methods Real-time PCR was used to detect the PML expression in peripheral blood mononuclear cells from 30 chronic myeloid leukemia patients and 24 healthy controls. Plasmids containing full length PML were constructed and transfected into K562 cell line to screen clones stably expressing PML, the clones stably expressing empty vector as a control. MTT assay and flow cytometry were used to detect the effect of PML on K562 cell proliferation and cell cycle. Furthermore, RT-PCR and western blot were used to detect c-myc and p27kipl （p27） mRNA and protein expression. Lastly, the mRNA levels of c-myc and p27 were detected in CML patients and healthy controls. Results The mRNA level of PML in CML patients （ACt =9.02±0. 74） was significantly lower than the healthy controls （ ACt = 7.91±0. 34） and the difference was statistically significant （ t = 5.07, P 〈 0.001 ） . PML over-expression could inhibit the proliferation of K562 cells obviously and cause GO/G1 cell cycle arrest. In addition, c-myc mRNA and protein expression decreased while p27 mRNA and protein expression increased in K562 cells with stably expressed PML compared with vector control. The mRNA level of c-mye in CML patients （△Ct = 7. 13±0. 43 ）was significantly higher than the healthy controls （△Ct = 9. 55±0. 82 ）and the difference was statistically significant（ t = 6. 78 ,P 〈 0. 001 ）, while the mRNA level of p27 in CML patients（△Ct = 4. 56±0. 58）was significantly lower than the healthy controls （△Ct = 3.29± 0. 92 ）and the difference was statistically significant（ t = 2. 93, P 〈 0. 01 ）. Conclusions PML protein is expressed highly in healthy controls, whereas its expression is declined significantly in chronic myeloid leukemia patients. PML may inhibit leukemia cell proliferation through regulating c-myc and p27 expression.