PMA诱导K562细胞向单核细胞分化机制探讨

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。

Signaling mechanism of PMA-induced monocytic differentiation of K562

OBJECTIVE： To explore the mechanism of PMA induce K562 cells differentiated to monocyte-macropha- ges. METHODS：The cell counting Kit 8（CCK8） assay was used to detect the effects of PMA on K562 cells proliferation. The cell morphology changes were observed by Wright-Giemsa staining. The cell surface markers CD11b,CD14 were ana- lyzed by flow cytometry. The expression of TGF-β1 ,SMAD3 ,and SMAD4 were detected by RT-PCR and the protein level of TGF-β1 was detected by western blot. RESULTS： PMA can inhibit the proliferation of K562 cells,and can promote the differentiation by dose dependent. The inhibition rates of 6.25,12.5,25,50,100 and 200 nmol/L PMA were （22.03± 2.7）%,（31.04±4.3）% ,（35. 03±3. 5）%,（47.01±4. 1）%,（55.06±5. 2）% and （76.72±5.4）% respectively（F= 2.24, P〈0.05）. Wright-Giemsa staining indicated that the numbers of mature monocytes were increased. The expression of the cell surface markers CD11b,CD14 was increased. In mRNA levels, the expression of TGF-β/SMAD signaling path- way factor TGF-β1 and its＇ downstream factors SMAD3,SMAD4 were obviously up-regulated. The expression of TGF-β1 was also up-regulated in protein levels. CONCLUSION： PMA may promote the differentiation of K562 cells by influencing the transduction of TGF-β/SMAD signaling pathway.