腺病毒介导的PML(NLS^-)对大黄素引起的HL-60细胞存活的影响及其机制

Effect of PML(NLS^-)Gene Mediated by Recobinant Adenovirus Vector on Emodin-induced Apoptosis of HL-60 Cells

目的探讨PML(NLS-)对大黄素引起的人HL-60细胞凋亡的影响及其机制。方法将构建成功的重组腺病毒Ad-PML(NLS-)和空载病毒Ad-KZ分别感染HL-60细胞,并设空白对照组。RT-PCR法和Western blot法检测各组细胞PML(NLS-)基因mRNA和蛋白的表达水平;MTT法分析腺病毒感染各组经60μmol/L大黄素处理后的细胞的增殖能力;各组感染HL-60细胞经60μmol/L大黄素处理后,FCM法分析细胞周期分布和凋亡率;并用RT-PCR法和Western blot法分别检测各组细胞凋亡相关基因C-MYC、BCL-2和BAX的mRNA及蛋白表达水平。结果与空白对照组和空载病毒组比较,RT-PCR和Western blot结果显示,AdPML(NLS-)组细胞内的PML(NLS-)表达水平明显增高。各组细胞经大黄素处理后,MTT结果显示,感染AdPML(NLS-)的HL-60细胞增殖能力明显提高;FCM结果显示,感染Ad-PML(NLS-)的HL-60细胞处于G1期细胞比例降低,S期细胞比例增高(P<0.05),且凋亡率降低(P<0.05);细胞内BCL-2和C-MYC基因mRNA的转录和蛋白表达水平增高,BAX基因mRNA的转录水平和蛋白表达水平均下降。结论过表达PML(NLS-)基因能够促进经过60μmol/L大黄素处理的HL-60细胞的增殖,可能通过上调BCL-2和C-MYC基因的表达、下调BAX基因的表达抑制细胞凋亡。

Objective To determine the effect and mechanism of action of PML(NLS-)gene on emodininduced apoptosis of human HL-60 cells.Methods HL-60 cells were infected with recombinant adenovirus AdPML(NLS-)and Ad-KZ,respectively.The PML(NLS-)gene was detected by Real-time PCR(RT-PCR)and Western blot.The proliferation level of the HL-60 cells was determined by MTT method.The HL-60 cells were treated with 60μmol/L emodin for 72 hand then analyzed by flow cytometry for their cell cycle and apoptosis rate.The transcription levels of apoptosis-related BCL-2,BAX and C-MYC genes were determined by RT-PCR.The translation levels of those genes were determined by Western blot.Results Compared with normal controls and the HL-60 cells infected with Ad-KZ,the mRNA and protein expression levels of PML(NLS-)gene increased significantly in the HL-60 cells infected with Ad-PML(NLS-).Increased proliferation levels of the Ad-PML(NLS-)infected HL-60 cells were observed in those treated with 60μmol/L emodin,which showed decreased percentage of cells at G1phase,increased percentage of cells at S phase,and decreased emodin-induced apoptosis.The levels of mRNA transcription and protein expression of BAXgene decreased,while those of BCL-2 and C-MYC genes increased significantly.Conclusion The over-expression of PML(NLS-)gene might promote the proliferation and arrest the apoptosis of HL-60 cells by up-regulating the expressions of BCL-2 and C-MYC genes and down-regulating the expression of BAXgene.