呼肠孤病毒导致白血病细胞凋亡的分子机制研究

Molecular Mechanisms of Apoptosis of Leukemia Cell Induced by Reovirus

目的初步探讨溶瘤病毒呼肠孤病毒3型(Reovirus 3,Reo3)致白血病HL60细胞凋亡的分子机制。方法用不同感染复数(MOI)的Reo3感染HL60细胞,设未感染的HL60细胞为对照组,病毒感染48 h后,通过CCK8法检测不同MOI的病毒对HL60细胞活性的影响;利用流式细胞术检测HL60细胞凋亡率;Western blot检测HL60细胞中双链RNA依赖性蛋白激酶(PKR)活化水平和凋亡相关蛋白表达情况。用PKR特异性抑制剂2-氨基嘌呤(2-AP)作用HL60细胞24 h后,Reo3感染HL60细胞48 h,检测细胞凋亡水平和凋亡相关蛋白表达水平。结果 MOI为1的Reo3作用于HL60细胞48 h后对细胞的抑制作用明显,细胞存活率为(24.333±3.396)%,细胞凋亡率为(29.96±2.06)%,与对照组比较差异均有统计学意义(P<0.05)。Western blot结果显示:与对照组比较,感染Reo3的HL60细胞PKR、 p-PKR、Bax、Caspase3、cleaved Caspase3蛋白表达均升高(P<0.05),而Bcl-2蛋白表达水平下降(P<0.05)。与未加抑制剂组比较,采用2-AP预处理的HL60细胞感染Reo3后细胞凋亡率下降(P<0.05),PKR磷酸化水平和凋亡相关蛋白表达水平亦下降(P<0.05)。结论溶瘤病毒Reo3作用HL60细胞可以引起PKR的活化,导致HL60细胞的凋亡。

Objective To investigate the molecular mechanism of apoptosis of HL60 cells induced by oncolytic virus Reovirus type 3(Reo3).Methods HL60 cells were infected with Reo3 at different multiplicity of infection(MOI)with the uninfected HL60 cells as control group.After 48 h of infection,the activity of HL60 cells infected with virus at different MOI was detected by CCK8 method to investigate the influence of MOI to cell activity.Simultaneously,the apoptotic rate of HL60 cells was detected by flow cytometry,and the activation level of double-stranded RNA-dependent protein kinase(PKR)and the expression of apoptotic-related protein in HL60 cells were detected by Western blot.Before infection with Reo3 for 48 h,HL60 cells were treated with 2-aminopurine(2-AP),a specific inhibitor of PKR,for 24 h.Afterward,the apoptotic level and expression of apoptotic related proteins were detected.Results Activity of HL60 cells was obviously inhibited after infected with Reo3 with a MOI of 1 for 48 h.The cell survival rate was(24.333±3.396)%and the apoptotic rate was(29.96±2.06)%.Both rates were all higher than those in the control group(P<0.05).Western blot results showed that the expression levels of PKR,p-PKR,Bax,Caspase3 and cleaved Caspase3 in HL60 cells infected with Reo3 were higher than those in the control group(P<0.05),while the expression level of Bcl-2 was lower(P<0.05).Compared with the group without inhibitor,the apoptotic rate of HL60 cells pretreated with 2-AP decreased(P<0.05),the phosphorylation level of PKR and the expression level of apoptotic-related protein also decreased(P<0.05).Conclusion Oncolytic virus Reo3 could activate PKR in HL60 cells and thus induce apoptosis of HL60 cells.