Fas抗原和caspase-8调控细菌氧化还原蛋白azurin诱导的人骨肉瘤细胞凋亡的实验研究

Modulation of bacterial redox protein azurin induces human osteosarcoma cell apoptosis by Fas antigen and caspase-8

目的:探讨Fas抗原和caspase-8在细菌氧化还原蛋白azurin诱导人骨肉瘤细胞凋亡中的调控机制及作用。方法:用150mg/L浓度的azurin处理U2OS细胞,分别作用6、12、24和48h。细胞免疫化学法检测Fas抗原的表达。用荧光试剂盒检测caspase-8的活性。流式细胞仪检测azurin和抗-Fas-抗体诱导的骨肉瘤细胞凋亡百分率,以及加入caspase-8活性抑制剂IETD-FMK后凋亡百分率的变化。结果:Azurin诱导U2OS细胞凋亡后,与对照组比较,Fas抗原表达强度增加(P<0.01),caspase-8活性升高(P<0.01)。Fas抗原表达和caspase-8活性随着azurin作用时间的延长而逐渐升高,至24h后达到高峰,然后缓慢下降,但仍显著高于对照组(P<0.01)。Fas抗原的表达与caspase-8活性变化呈显著正相关(r=0.873,P<0.01)。表达增强的Fas抗原具有转导凋亡信号的功能,抗-Fas抗体借此增强azurin诱导U2OS细胞凋亡的作用。Caspase-8活性抑制剂IETD-FMK能阻断caspase-8活化而抑制azurin或抗-Fas抗体诱导的U2OS细胞凋亡,与抑制剂组比较,细胞凋亡百分率显著差异(P<0.01)。结论:Fas依赖途径可能为细菌氧化还原蛋白azurin诱导U2OS细胞凋亡主要机制之一,caspase-8活性上调可能在凋亡过程中发挥重要作用。

AIM： To determine the role of Fas antigen and caspase -8 in modulating apoptosis of osteosarcoma cells induced by bacterial redox protein azurin. METHODS： The human osteosarcoma cell line U20S was treated with bacterial redox protein azurin （ 150 mg/L） for 6, 12, 24 and 48 h, respectively. Cell immunohistochemistry and quantitative image pattern analysis were applied for detecting the expression of Fas antigen. Caspase - 8 activity was detected using caspase - 8 fluorescent assay kit. The apoptotic rate was measured by FCM. RESULTS： Compared with the control group, the expression of Fas antigen and activity of caspase - 8 significantly increased in U2OS cells treated with 150 mg/L azurin （ P 〈 0.01 ）. There was positive correlation between the expression of Fas antigen and activity of caspase- 8 （ r =0.873, P 〈 0.01）. The increased Fas antigen expression had the function to transfer apoptotic signals and the anti - Fas antibody promoted azurin induced apoptosis through increasing Fas antigen expression. IETD - FMK blocked the activation of procaspase - 8 and reduced apoptosis of U2OS cells in the presence of azurin or anti- Fas antibody. The apoptotic rates in azurin group and anti- Fas antibody group were significantly different from the inhibitor group （ P 〈 0.01 ）. CONCLUSION： The Fas - dependent signalling is the important pathway of azurin inducing apoptosis in U2OS cells. The up - regulation of csepase - 8 may play an important role.