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目的:研究高三尖杉酯碱(homohar-ringtonine,HHT)诱导HL-60细胞分化后CD44表达变化及其与细胞周期调控的关系,探讨HHT的作用机制。方法:建立HHT诱导分化模型,瑞氏染色观察细胞形态,MTT法检测细胞增殖变化,NBT还原实验测定细胞分化状态,流式细胞术检测细胞表面CD11b表达及细胞周期变化,RT-PCR检测CD44、p27、p21和Cyclin E基因在mRNA水平的表达变化。结果:MTT实验显示,HHT对HL-60细胞产生增殖抑制作用,HL-60细胞G0/G1期细胞百分比明显上升,S期细胞百分比明显下降,P<0.05。半定量分析PCR扩增产物光密度相对表达量显示,HHT诱导后HL-60细胞中CD44基因表达逐渐减弱,p27和p21基因逐渐增强,Cyclin E基因逐渐减弱。CD44与p27表达呈明显负相关,r=-0.686,P<0.05。结论:HHT可能通过下调CD44基因,进而提高p27和p21表达,抑制Cyclin E活性而对HL60细胞产生诱导分化作用。
OBJECTIVE: To study the relationship between CD44 expression and cell cycle regulation after homohar-ringtonine (HHT) inducing HL-60 cells differentiation and to explore the mechanism of HHT. Methods: The HHT induced differentiation model was established. Wright’s stain was used to observe the cell morphology. MTT assay was used to detect the cell proliferation. Cell differentiation was determined by NBT reduction assay. CD11b expression and cell cycle were detected by flow cytometry. CD44, The expression of p27, p21 and Cyclin E at mRNA level. Results: MTT assay showed that HHT could inhibit the proliferation of HL-60 cells. The percentage of cells in G0 / G1 phase of HL-60 cells increased significantly and the percentage of cells in S phase decreased significantly (P <0.05). Semi-quantitative analysis of the relative optical density of PCR products showed that the expression of CD44 gene in HL-60 cells gradually decreased, the p27 and p21 genes gradually increased, and the Cyclin E gene gradually decreased after HHT induction. The expression of CD44 was negatively correlated with the expression of p27, r = -0.686, P <0.05. Conclusion: HHT may induce differentiation of HL60 cells by down-regulating CD44 gene, increasing p27 and p21 expression and inhibiting Cyclin E activity.