调控A20对NF-κB表达及Jurkat细胞的影响

Effect of Regulating A20 Expression on NF-κB Expression and Biological Characteristics of Jurkat Cells

目的:探讨在急性T淋巴细胞白血病糖皮质激素耐药Jurkat细胞株中,调控A20对NF-κB表达以及Jurkat细胞生物学特性的影响。方法:CCRF CEM和Jurkat细胞中分别加入100、10、1、0.1、0.01和0.001μmol/L地塞米松(DEX),培养24、48和72 h,应用CCK-8检测细胞增殖抑制的变化。构建A20质粒,设计并合成A20-siRNA,分别通过脂质体转染Jurkat细胞,CCK-8检测不同浓度DEX处理组、不同浓度DEX联合A20质粒和A20-siRNA处理组的Jurkat细胞增殖率。应用RT-qPCR检测NF-κB的m RNA表达水平,Western bolt检测蛋白表达水平,采用流式细胞术检测Jurkat细胞凋亡。结果:不同浓度地塞米松(DEX)对CCRF CEM细胞生长的抑制作用呈显著的时间依赖性(r=0.984,P<0.05)和浓度依赖性(r=0.966,P<0.05);CCRF CEM细胞在24 h时,IC50接近1μmol/L,Jurkat细胞在1μmol/L和10μmol/L开始出现较大差异。由于1μmol/L DEX处理的Jurkat细胞增殖率无明显变化,故选取1μmol/L DEX处理的Jurkat细胞为对照组。检测显示A20质粒转染联合不同浓度DEX处理组细胞增殖率较DEX组及A20-siRNA联合DEX组低(P<0.05)。A20质粒转染Jurkat细胞后,NF-κB的mRNA及蛋白表达明显低于对照组(P<0.05),细胞凋亡率较对照组明显增高(P<0.05);A20-siRNA转染Jurkat细胞后,NF-κB的mRNA及蛋白表达水平明显高于对照组(P<0.05),A20-siRNA组细胞的凋亡率与对照组无明显改变(P>0.05)。结论:Jurkat细胞对DEX耐药,A20过表达联合DEX可增加对DEX耐药细胞的敏感性,降低Jurkat细胞增殖率;下调Jurkat细胞中NF-κB的表达水平,促进Jurkat细胞的凋亡。

Objective:To explore the effect of regulating A20 expression on NF-κB and biological characteristics of Jurkat cells with glucocorticoid(GC)resistance.Methods:CCRF CEM and Jurkat cells were treated with dexamethasone(DEX)at concentrations of 100、10、1、0.1、0.01 and 0.001μmol/L,and cultured for 24、48 and 72 h.The proliferation inhibition rate of Jurkat cell was detected by CCK-8.A20 plasmid was constructed,A20-siRNA was designed and synthesized,and transfected into Jurkat cells by liposome.CCK-8 was used to detect the proliferation rates of Jurkat cells in different concentrations of DEX group,DEX combined with A20 plasmid group and A20-siRNA group.The mRNA expression level of NF-κB was detected by RT-qPCR,the protein expression level of NF-κB was detected by Western blot,and the apoptosis of Jurkat cells was examined by flow cytometry.Results:The inhibitory effects of DEX at different concentrations on the growth of CCRF CEM cells were time-dependent(r=0.984,P<0.05)and concentration-dependent(r=0.966,P<0.05).At the point of 24 hour,the IC50 approached 1μmol/L in CCRF CEM cells.Great large differences began to appear between 1 and 10μmol/L,the proliferation rate of Jurkat cells treated with 1μmol/L DEX did not show a significant change.Therefore,1μmol/L was selected as control group.The cell proliferation rate of A20 plasmid transfection combined with different concentrations of DEX group was lower than that of DEX group and A20-siRNA combined with DEX group.After transfection of A20 plasmid,the expression level of NF-κB was significantly lower than that of control group(P<0.05),and the apoptotic rate was significantly higher than that of control group(P<0.05).After transfection of Jurkat cells with A20-siRNA,the expression level of NF-κB was significantly higher than that of control group(P<0.05).The apoptotic rate of cells in A20-siRNA group was not significantly changed(P>0.05).Conclusion:Jurkat cells are resistant to DEX.A20 overexpression combined with DEX can increase sensitivity of Jurkat cells with GC resistance and decrease the proliferation rate of Jurkat cells,down-regulate the expression level of NF-κB and promote the apoptosis of Jurkat cells.