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本研究探讨中药补骨脂素(psoralen,PSO)加长波紫外线A(ultraviolet A,UVA)(PUVA)诱导人白血病细胞HL-60凋亡的作用及其可能的作用机制。采用MTT法观察PUVA对HL-60细胞增殖的影响;采用电子显微镜技术观察细胞超微结构改变;流式细胞术(FCM)检测细胞凋亡率、线粒体跨膜电位水平以及细胞Fas、FasL蛋白的表达;荧光定量PCR技术检测细胞Fas、FasL mRNA的表达;免疫细胞化学法(immunocytochemistry,ICC)检测caspase8、caspase 3蛋白的表达。结果表明,PUVA可抑制HL-60细胞的增殖,使凋亡率增加,作用呈时间、浓度依赖性;UVA照射时间15分钟和PSO浓度为80μg/ml时,HL-60细胞增殖的抑制率、凋亡率达峰值;PUVA作用后HL-60细胞超微结构出现明显的凋亡形态学改变,细胞线粒体跨膜电位水平下降;PUVA作用4小时Fas mRNA的表达升高,FasL mRNA的表达下降;PUVA作用24小时Fas、FasL在蛋白水平的表达亦呈现相同规律;PUVA可使HL-60细胞caspase 8、caspase 3蛋白的表达增强,在作用后8小时强度达峰值。结论:PUVA能够抑制HL-60细胞的增殖,并诱导其凋亡,可能的机制是PUVA作用于Fas/FasL系统,使Fas基因表达升高、FasL基因表达下降,激活下游caspase 8、caspase 3的表达,线粒体膜电位水平降低亦可能参与了这个过程。
This study was designed to investigate the effects of psoralen (PSO) and ultraviolet A (UVA) on the apoptosis of human leukemia cells HL-60 and its possible mechanism. The effects of PUVA on the proliferation of HL-60 cells were observed by MTT assay. The ultrastructural changes of HL-60 cells were observed by electron microscopy. The apoptosis rate, the transmembrane potential of mitochondria and the expression of Fas and FasL proteins were detected by flow cytometry (FCM) The expression of Fas and FasL mRNA was detected by real-time PCR. The expressions of caspase8 and caspase 3 were detected by immunocytochemistry (ICC). The results showed that PUVA could inhibit the proliferation of HL-60 cells and increase the apoptosis rate in a time-and concentration-dependent manner. Inhibition of proliferation of HL-60 cells by UVA irradiation at 15 min and PSO concentration of 80 μg / The apoptosis rate of HL-60 cells reached the peak value after PUVA treatment. The ultrastructural changes of HL-60 cells were observed and the mitochondrial transmembrane potential was decreased. The expression of Fas mRNA and FasL protein were increased at 4 hours after PUVA treatment. The expression of Fas and FasL at protein level also showed the same pattern at 24 h after PUVA treatment. PUVA enhanced the expression of caspase 8 and caspase 3 in HL-60 cells, and peaked at 8 h. CONCLUSION: PUVA can inhibit the proliferation and induce the apoptosis of HL-60 cells. The possible mechanism is that PUVA acts on Fas / FasL system, which leads to the increase of Fas gene expression and the decrease of FasL gene expression, and the activation of downstream caspase 8 and caspase 3 Expression, mitochondrial membrane potential reduction may also be involved in this process.