Objective: To investigate the effects of knocking down nucleostemin ( NS) combined with rapamycin (RAPA) on autophagy and apoptosis in HL-60 cells , and to explore its role in HL-60 cells .
Methods: The expression of NS protein was detected using Western blot , after transfection of HL-60 cells was achieved by the recombinant lentviral vector NS -RNAi-GV248 . Flow cytometry was used to detect changes in cells apoptosis after NS silencing/ rapamycin for 24 , 48 hours , and the expressions of NS , LC3 , p62 , BCL-2 and Bax proteins in cells were detected by Western blot.
Results: The expression of NS in HL-60 cells was successfully down-regulated by recombinant lentiviral vector. After treatment with rapamycin for 24 and 48 h , the apoptosis rate of cells in each group increased (P < 0.05) , and the apoptosis was more obvious at 48 hours . Compared with the NS silencing group or rapamycin group , after treated with NS down-regulation combined with rapamycin for 48 hours , the apoptosis of HL-60 cells was significantly increased ( P < 0.05 ) , LC3 -II/LC3 -I ratio was significantly increased ( P < 0.05 ) , p62 protein expression was significantly decreased (P < 0.05) , and BCL-2/Bax ratio was significantly decreased ( P < 0.05) .
Conclusion: NS down-regulation combined with rapamycin can enhance the apoptosis and autophagy of HL-60 cells , and the induction of apoptosis of HL-60 cells may be related to the expression of BCL-2 and Bax proteins .
题目: Nucleostemin 下调联合雷帕霉素对 HL-60 细胞自噬 与凋亡影响的初步研究.
目的: 研究 Nucleostemin (NS)下调联合雷帕霉素对 HL-60 细胞自噬和凋亡的影响并探讨其在 HL-60 细胞 中的作用。.
方法: 通过 NS-RNAi-GV248 重组慢病毒载体转染 HL-60 细胞后检测 NS 蛋白的表达。采用流式细胞术 检测沉默 NS 和／或雷帕霉素处理 24 、48 h 后 HL-60 细胞的凋亡变化；采用 Western blot 技术检测各组细胞中 NS、 LC3 、p62 、BCL-2 、Bax 蛋白的表达。.
结果: 重组慢病毒载体成功下调 HL-60 细胞 NS 表达。经雷帕霉素处理后，细胞 凋亡率均明显增加(P <0.05) , 且 48 h 凋亡更明显。与 NS 敲低组、雷帕霉素组相比，NS 下调联合雷帕霉素组处理 48 h 后，其凋亡明显增加(P <0 . 05) , LC3 -II/LC3 -I 比值显著增加(P < 0.05) , p62 蛋白表达下降更显著(P < 0.05) , 并且 BCL-2/Bax 比值减低更明显(P < 0.05) 。.
结论: NS 下调联合雷帕霉素可增强 HL-60 细胞的凋亡和自噬，并且 其诱导 HL-60 细胞的凋亡可能与 BCL-2 、Bax 蛋白的表达有关。.
Keywords: HL-60 cells; nucleostemin; apoptosis; autophagy; rapamycin.