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目的:探讨肿瘤坏死因子相关诱导凋亡配体(TRAIL)与蛋白酶抑制剂MG132联合诱导细胞凋亡的可能机制。方法:倒置显微镜下观察TRAIL、蛋白酶抑制剂MG132及两药联合干预24 h A549细胞形态学变化;流式细胞仪技术检测TRAIL、蛋白酶抑制剂MG132及两药联合干预24 h A549细胞凋亡指数变化;ELISA方法检测TRAIL、蛋白酶抑制剂MG132及两药联合干预24 h A549细胞NF-κB活性变化;蛋白印迹技术检测TRAIL、蛋白酶抑制剂MG132及两药联合干预24 h A549细胞凋亡基因蛋白Bax及Caspase-3表达水平。结果:倒置显微镜下可观察到两药联合组细胞呈现皱缩、核碎裂等典型的凋亡特征。流式细胞仪技术结果显示TRAIL与蛋白酶抑制剂MG132联合用药干预24 h细胞凋亡率约为48.78%,与单药组(21.12%)相比,凋亡率显著提高,P=0.002;ELISA结果显示TRAIL与MG132联合干预24 h后,细胞NF-κB活性约为0.23,与TRAIL单药组相比活性明显降低,P=0.008;蛋白印迹技术结果显示TRAIL与蛋白酶抑制剂MG132联合用药干预24 h细胞凋亡基因蛋白Bax及Caspase-3表达显著升高,与单药组相比,具有统计学意义,P=0.005。结论:TRAIL联合蛋白酶抑制剂MG132能显著提高细胞的凋亡率,其机制可能与两药联合后降低NF-κB活性、促进凋亡基因蛋白Bax的表达,进而增高Caspase-3表达有关。
Objective: To investigate the possible mechanism of apoptosis induced by tumor necrosis factor related apoptosis inducing ligand (TRAIL) and proteasome inhibitor MG132. Methods: The morphological changes of TRAIL, MG132 and 24 h A549 cells were observed under inverted microscope. The apoptosis index of TRAIL, MG132 and 24 h A549 cells were detected by flow cytometry ; ELISA assay TRAIL, protease inhibitor MG132 and two drugs combined intervention 24 hours A549 cells NF-κB activity changes; Western blotting detection of TRAIL, proteasome inhibitor MG132 and two drugs combined intervention 24 h A549 apoptosis gene protein Bax and Caspase-3 expression level. Results: Under the inverted microscope, typical apoptotic features such as crinkles and nuclear fragmentation were observed in the two groups. The results of flow cytometry showed that the apoptotic rate of TRAIL treated with MG132 for 24 h was 48.78%, which was significantly higher than that of monotherapy group (21.12%) (P = 0.002). ELISA results The results showed that the activity of NF-κB in TRAIL combined with MG132 cells for 24 h was about 0.23, which was significantly lower than that of TRAIL alone group (P = 0.008). Western blotting showed that the combination of TRAIL and MG132 interfered for 24 h The expression of Bax and Caspase-3 were significantly increased, which was statistically significant compared with single drug group (P = 0.005). CONCLUSION: TRAIL combined with protease inhibitor MG132 can significantly increase the apoptosis rate of cells, which may be related to the decrease of NF-κB activity, the increase of the expression of Bax and the increase of Caspase-3 expression after the two drugs combined.