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目的:探讨HIF-1α对电离辐射诱导人非霍奇金淋巴瘤(NHL)细胞凋亡的作用及其调控机制。方法:以HIF-1α抑制剂Echinomycin(EC)预处理人NHL细胞后给予5Gy X线照射,采用AnnexinⅤ染色方法检测肿瘤细胞凋亡水平的变化,采用蛋白质印迹法检测各细胞系中凋亡相关蛋白Bax、Bcl-2和Caspase-3的表达水平。将HIF-1αsiR-NA反义转染至NHL细胞中,检测转染后细胞凋亡分数及Caspase-3蛋白表达水平。结果:流式细胞仪检测结果显示,Namalwa细胞空白对照组、单纯照射组(RI)、RI+EC 2nmol/L组的凋亡率分别为5.27±1.13、13.81±3.12和39.96±6.81,Ramos细胞分别为6.02±1.26、12.98±3.07和40.76±11.58,Raji细胞则为7.16±0.46、17.62±4.94和47.85±10.22,P<0.01。电离辐射后,转染vector和HIF-1αsiRNA的Namalwa细胞凋亡率分别为15.32±3.64和20.20±1.87,Ramos细胞分别为15.05±2.46和21.02±3.12,t值分别为3.99和3.19,P<0.05;而Raji细胞则为10.90±1.65和17.26±0.69,t=5.98,P<0.01。蛋白质印迹法结果显示,通过EC预处理抑制HIF-1α的表达能够明显上调射线诱导的各NHL细胞系中Caspase-3蛋白表达水平,同时凋亡相关蛋白Bcl-2表达减低而Bax蛋白表达增加,Bcl-2/Bax比值明显降低,与单纯照射组相比差异均有统计学意义,P<0.05。结论:抑制HIF-1α能够增加射线诱导的NHL细胞凋亡,其机制可能与增加Bax蛋白表达而下调Bcl-2蛋白表达有关。
Objective: To investigate the effect of HIF-1α on apoptosis in human non-Hodgkin’s lymphoma (NHL) induced by ionizing radiation and its regulatory mechanism. Methods: Human NHL cells were pretreated with HIF-1α inhibitor Echinomycin (EC) and irradiated with 5Gy X-rays. The apoptosis of tumor cells was detected by AnnexinⅤ staining. The apoptosis-related proteins Bax, Bcl-2 and Caspase-3 expression levels. Antisense HIF-1αsiR-NA was transfected into NHL cells, and the apoptotic score and the expression of Caspase-3 protein were detected. Results: The results of flow cytometry showed that the apoptosis rates of Namalwa cells in blank control group, RI group and RI + EC 2nmol / L group were 5.27 ± 1.13, 13.81 ± 3.12 and 39.96 ± 6.81, respectively. Ramos cells 6.02 ± 1.26, 12.98 ± 3.07 and 40.76 ± 11.58 respectively, while the Raji cells were 7.16 ± 0.46, 17.62 ± 4.94 and 47.85 ± 10.22 respectively (P <0.01). After ionizing radiation, the apoptosis rates of Namalwa cells transfected with vector and HIF-1αsiRNA were 15.32 ± 3.64 and 20.20 ± 1.87 respectively, Ramos cells were 15.05 ± 2.46 and 21.02 ± 3.12 respectively, the t values were 3.99 and 3.19, P <0.05 While Raji cells were 10.90 ± 1.65 and 17.26 ± 0.69, t = 5.98, P <0.01. Western blotting showed that the inhibition of HIF-1α expression by EC pretreatment significantly up-regulated the expression of Caspase-3 protein in radiation-induced NHL cell lines, while decreased the expression of Bcl-2 and Bax protein, The ratio of Bcl-2 / Bax was significantly lower than that of the control group (P <0.05). CONCLUSION: Inhibition of HIF-1α can increase the radiation-induced apoptosis of NHL cells. The mechanism may be related to increasing Bax protein expression and down-regulating Bcl-2 protein expression.