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目的探讨西罗莫司(SRL)在体外诱导人肝癌细胞株BEL-7402的凋亡作用及其机制。方法以不同浓度的SRL(5、10、20、30、40和50nmol/L)作用于体外培养的人肝癌细胞株BEL- 7402,应用流式细胞仪检测培养24、48和72h时的细胞凋亡情况；Hoechst 33258荧光染色法观察细胞凋亡时的形态学变化；Western Blot法观察BEL-7402细胞中Caspase-3、Bcl-2、Bcl-xl和Bax基因的表达变化；采用Caspase-3试剂盒检测Caspase-3酶的活性；JC-1染色法检测细胞线粒体膜电位(Γψm)。结果SRL可诱导BEL-7402细胞凋亡,并与药物浓度和作用时间呈正相关。SRL作用BEL-7402细胞后48h,在Hoechst 33258荧光染色图片上可见核浓缩及核碎裂等典型的细胞凋亡特征,凋亡过程中线粒体膜电位下降及Caspase-3酶原蛋白激活,伴有Bcl-2、Bcl-xl蛋白表达的降低和Bax蛋白上调。结论SRL能使抗凋亡蛋白Bcl-2、Bcl-xl表达降低,促凋亡蛋白Bax表达上调,线粒体膜电位下降,激活Caspase-3酶原蛋白,从而诱导细胞凋亡。
Objective To investigate the apoptosis of human hepatocellular carcinoma cell line BEL-7402 induced by sirolimus and its mechanism. Methods The human hepatocellular carcinoma cell line BEL-7402 was treated with different concentrations of SRL (5, 10, 20, 30, 40, and 50 nmol / L). Flow cytometry was used to detect the apoptosis of cells at 24, 48 and 72 h Hoechst 33258 fluorescence staining was used to observe the morphological changes of apoptotic cells. Western Blot was used to detect the expression of Caspase-3, Bcl-2, Bcl-xl and Bax genes in BEL-7402 cells. Caspase- Caspase-3 activity was detected by Caspase-3 assay, and mitochondrial membrane potential (Γψm) was detected by JC-1 staining. Results SRL could induce the apoptosis of BEL-7402 cells and was positively correlated with the drug concentration and time. Typical apoptotic features such as nuclear condensation and nuclear fragmentation were observed on Hoechst 33258 fluorescence staining 48 hours after BEL-7402 cells were treated with SRL. Mitochondrial membrane potential and apoptosis of caspase-3 were associated with Bcl-2, Bcl-xl protein expression decreased and Bax protein upregulation. Conclusion SRL can decrease the expression of anti-apoptotic proteins Bcl-2 and Bcl-xl, increase the expression of pro-apoptotic protein Bax, decrease the mitochondrial membrane potential and activate Caspase-3 proenzyme, thereby inducing apoptosis.