沉默NRP-1基因对人T细胞白血病Jurkat细胞株增殖与凋亡的影响

Effects on proliferation and apoptosis of T-cell leukemia cells by silencing NRP-1

目的:探讨RNAi沉默NRP-1基因对人T细胞白血病细胞株增殖与凋亡的影响。方法:将我们前期构建好的p LB-NRP-1/shRNA重组慢病毒质粒,感染至Jurkat细胞中,用CCK-8法检测各组细胞的增殖情况及化疗药物表柔比星(EPI)处理后细胞增殖情况;用AV/PI法结合流式细胞仪检测细胞凋亡、细胞周期。结果:细胞增殖结果:在48、72、96 h时间点NRP-1/shRNA干扰组的OD值均低于相应对照组,差异有统计学意义(P<0.05),而阴性对照组与空白对照组差异无统计学意义(P>0.05);细胞凋亡率结果:与对照组比较,NRP-1/shRNA干扰组细胞的凋亡率明显升高,差异有统计学意义(P<0.05);对化疗药物表柔比星(EPI)敏感性检测结果:EPI浓度为0.025、0.05、0.1、0.2、0.4μg/ml时,NRP-1/shRNA干扰组的细胞生长抑制率较相应对照组高,差异皆有统计学意义(P<0.05),并且选择IC50进行EPI诱导后,NRP-1/shRNA干扰组的细胞凋亡率明显升高,与对照组比较差异有统计学意义(P<0.05);WB结果显示:与对照组相比,NRP-1/shRNA干扰组Bcl-2蛋白的表达水平明显下降,Bax的表达水平明显升高,差异均具有统计学意义(P<0.05);细胞周期结果:与对照组比较,NRP-1/shRNA干扰组G0/G1期细胞的比例增高,S期细胞的比例明显降低,差异均有统计学意义(P<0.05)。结论:RNAi沉默NRP-1基因可抑制Jurkat细胞增殖,促进其凋亡,提高对化疗药物的敏感性,其机制可能涉及Bcl-2/Bax途径的调节;并可将细胞周期阻滞在G0/G1期,降低细胞的增殖水平,诱导细胞进入凋亡期。

Objective： To investigate the effect on proliferation and apoptosis of T-cell leukemia cells by silencing NRP-1（ Jurkat cells）. Methods： The lentivirus plasmid which expresses NRP1 gene specific shRNA was constructed in our preliminary experimental. We transfected the lentivirus plasmid to human T-cell Lymphoma cells. The proliferation of Jurkat cells different groups and effect on cell proliferation after chemotherapy drug EPI-treated were found by CCK-8 kit. The proliferation level and apoptosis rate of the cells were detected by flow cytometry and Annexin-V-FITC/PI method. Results： The proliferation level of NRP-1/shRNA interference group was decreased significantly in 48 h,72 h,96 h,which was compared with the control groups. The apoptosis rate of the NRP-1 /shRNA interference group was increased compared with control groups. The chemotherapy drug sensitivity of epirubicin（ EPI） test results showed that EPI concentration was 0. 025,0. 05,0. 1,0. 2,0. 4 μg/ml,the NRP-1/shRNA interference group of cell growth inhibition rate was increased,the corresponding control group difference had statistical significance（ P〈0. 05）. We choose the drug concentration of the EPI IC50 for next experiments. NRP-1/shRNA interference group cell apoptosis rate increased significantly after induction,compared with the control groups difference was statistically significant（ P〈0. 05）. Compared with control group,the expression level of Bcl-2 protein was decreased and the expression level of bax protein was increased significantly after EPI induction. The percentage of cells at G0/G1 phase increased significantly,while those at S phase decreased significantly. Conclusion： Plasmid shRNA-NRP1 inhibited the expression of NRP1 in Jurkat cells and decreased the proliferation level of Jurkat cells and promote their apoptosis and enhance their drug sensitivity; the molecular mechanism may relate to down-regulation of Bcl-2 and up-regulation of Bax. and arrested the cell cycle at G0/G1 phase.