HL-60细胞高三尖杉酯碱诱导后CD44表达变化及机制的研究

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。

Study on variation of CD44 expression and its role in differentiation of HL-60 cells induced by HHT

OBJECTIVE: To study the expression of CD44 and its role in HL-60 cells induced by HHT.METHODS:HHT inducing model was established.Cell morphous was observed by Wright's staining.Cell proliferation was detected by MTT assay.Cell differentiation was indicated by NBT reduction assay and CD11b positive percent and cell cycle were detected by flow cytometry.The mRNA expressions of CD44,p27,p21 and Cyclin E were detected by RT-PCR assay.RESULTS: The proliferation of HL60 cells was obviously inhibited by HHT,which was accompanied with the up-regulation of differentiation,with the increase of G0/G and decrease of S phase,P<0.05.The results of RT-PCR assay indicated that the expressions of CD44 and Cyclin E were down-regulated and the expressions of p27 and p21 were up-regulated.Furthermore,there was a negative correlation between the expressions of CD44 and p27 mRNA,r=-0.686,P<0.05.CONCLUSION:HHT plays an important role in the differentiation of HL-60 cells,which is due to the down-regulation of CD44 and Cyclin E,and up-regulation of p27 and p21.