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目的:探讨佛波酯(phorbol-12-myristate-13-ace-tate,PMA)诱导K562细胞向单核/巨噬细胞分化的作用机制。方法:采用CCK8法检测0、6.25、12.5、25、50、100和200nmol/L PMA对K562细胞增殖的影响,应用Wright-Gimesa染色观察细胞形态学变化,采用流式细胞术检测细胞表面分化抗原CD11b和CD14的表达变化,采用RT-PCR分析TGF-β1及其下游基因SMAD3和SMAD4在基因水平的表达趋势,并采用蛋白质印迹法检测TGF-β1在蛋白水平的表达变化。结果:K562细胞经6.25nmol/L PMA处理后72h增殖抑制率为(22.03±2.7)%,12.5nmol/L为(31.04±4.3)%,25nmol/L为(35.03±3.5)%,50nmol/L为(47.01±4.1)%,100nmol/L为(55.06±5.2)%,200nmol/L为(76.72±5.4)%,差异有统计学意义,F=2.24,P<0.05。PMA能抑制K562细胞的增殖并能促进其分化,作用效果随剂量的加大逐渐增强;细胞形态学上趋向于成熟分化;细胞表面分子CD11b和CD14的表达量升高。在mRNA水平,TGF-β/SMAD信号通路因子TGF-β1的表达量随时间的延长逐渐升高其下游因子SMAD3和SMAD4的表达也呈上升趋势;在蛋白水平,TGF-β1的表达亦呈上升趋势。结论:PMA可通过促进TGF-β/SMAD信号的传导诱导白血病细胞株K562细胞向单核/巨噬细胞分化。
Objective: To investigate the mechanism of phorbol-12-myristate-13-a-tate (PMA) inducing K562 cells to differentiate into monocytes / macrophages. Methods: The effects of 0, 6.25, 12.5, 25, 50, 100 and 200 nmol / L PMA on the proliferation of K562 cells were detected by CCK8 assay. The cell morphology was observed by Wright-Gimesa staining. Flow cytometry was used to detect cell surface differentiation antigen The expression of TGF-β1 and its downstream genes SMAD3 and SMAD4 at gene level were analyzed by RT-PCR. The expression of TGF-β1 at the protein level was detected by Western blotting. Results: The proliferation inhibition rates of K562 cells treated with 6.25nmol / L PMA for 72 hours were (22.03 ± 2.7)%, (31.04 ± 4.3)% for 12.5nmol / L and 35.03 ± 3.5% for 25nmol / L (47.01 ± 4.1)%, 100 nmol / L was (55.06 ± 5.2)% and 200 nmol / L was (76.72 ± 5.4)%, the difference was statistically significant, F = 2.24, P <0.05. PMA could inhibit the proliferation and promote the differentiation of K562 cells. The effect of PMA gradually increased with the increase of dosage. The morphology of MMA cells tended to mature and differentiation. The expression of CD11b and CD14 on cell surface increased. At the mRNA level, the expression of TGF-β1 and TGF-β1 in TGF-β / SMAD signaling pathway gradually increased with the prolongation of time, and the expression of downstream factors SMAD3 and SMAD4 also showed an upward trend. At the protein level, the expression of TGF-β1 also increased trend. Conclusion: PMA can induce leukemia cell line K562 cells to differentiate into monocytes / macrophages by promoting TGF-β / SMAD signal transduction.