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目的:研究iASPP基因全长(iASPP-FL)及iASPP基因短型剪接体(iASPP-SV)对肿瘤细胞凋亡及基因组不稳定性的影响。方法:用iASPP-FL及iASPP-SV表达载体转染MCF-7细胞,筛选稳定表达目标蛋白的单克隆细胞株,分别进行137铯照射及依托泊苷处理,流式细胞仪检测细胞凋亡水平,并通过碱性单个细胞凝胶电泳(彗星实验)及H2AX蛋白表达水平变化分析细胞是否存在基因组不稳定性。结果:稳定表达iASPP-FL及iASPP-SV的MCF-7细胞株在受到200μg/mL Vp-16处理后,相对于转染空载体组的早期凋亡率为(42.70±2.38)%,晚期凋亡率为(28.94±1.38)%,SV1和SV2细胞早期凋亡率分别下降为(24.40±1.12)%和(17.52±1.08)%,晚期凋亡率分别下降为(18.78±0.95)%和(19.58±1.04)%;但是H2AX蛋白水平升高分别达到转染空载体组的1.48、2.10倍。FL1和FL2细胞早期凋亡率分别为(14.22±0.68)%和(15.82±0.74)%,晚期凋亡率分别为(11.16±0.88)%和(9.88±0.56)%,均比转染空载体组的(31.22±1.64)%和(36.90±2.08)%明显下降;H2AX蛋白水平升高分别达到转染空载体组的2.09和1.99倍。4Gy 137铯照射后,SV1和FL1细胞早期凋亡率分别为(6.42±0.24)%和(7.05±0.24)%,细胞晚期凋亡率分别为(15.10±0.50)%和(15.14±0.58)%,均显著低于转染空载体组的早期凋亡率(16.56±0.81)%和晚期凋亡率(29.08±1.55)%。彗星实验也显示,在遭受DNA损伤后iASPP-FL及iASPP-SV细胞株比对照组存在更加明显的彗星拖尾现象。结论:iASPP基因可以抑制MCF-7细胞受到放射和药物作用后发生的凋亡,但是细胞DNA双链断裂损伤增加,细胞群体中保留DNA损伤的细胞比例增加,可能会导致基因组不稳定性持久存在并累积,导致恶性肿瘤的发生发展。
Objective: To study the effects of iASPP-FL and iASPP-SV on apoptosis and genomic instability of tumor cells. METHODS: MCF-7 cells were transfected with iASPP-FL and iASPP-SV expression vector, and the monoclonal cell lines stably expressing the target protein were screened. The cells were treated with 137 cesium and etoposide respectively, and the apoptosis levels were detected by flow cytometry The cell instability was analyzed by alkaline single cell gel electrophoresis (comet assay) and H2AX protein expression level. Results: The apoptosis rate of MCF-7 cells stably expressing iASPP-FL and iASPP-SV after treatment with 200μg / mL Vp-16 was (42.70 ± 2.38)%, compared with that of the control group The apoptotic rates of SV1 and SV2 cells were (24.40 ± 1.12)% and (17.52 ± 1.08)% respectively, and the rates of late apoptosis were (18.78 ± 0.95)% and 19.58 ± 1.04)% respectively. However, the H2AX protein level was 1.48 and 2.10 times higher than that of the blank vector transfected group respectively. The early apoptotic rates of FL1 and FL2 cells were (14.22 ± 0.68)% and (15.82 ± 0.74)% respectively, and the late apoptotic rates were (11.16 ± 0.88)% and (9.88 ± 0.56)%, respectively, (31.22 ± 1.64)% and (36.90 ± 2.08)%, respectively. The levels of H2AX protein were 2.09 and 1.99 times higher than that of the blank vector transfected group respectively. The early apoptotic rates of SV1 and FL1 cells were (6.42 ± 0.24)% and (7.05 ± 0.24)%, respectively, and the late apoptotic rates were (15.10 ± 0.50)% and (15.14 ± 0.58)%, respectively, (16.56 ± 0.81)% and the late apoptosis rate (29.08 ± 1.55)%, respectively. Comet assay also showed that there was more obvious comet tailing in iASPP-FL and iASPP-SV cell lines than control group after DNA damage. Conclusion: The iASPP gene can inhibit the apoptosis of MCF-7 cells after radiation and drug treatment. However, DNA double-strand breaks increase in cell population, and the proportion of cells with DNA damage in the cell population may increase, which may result in persistent genomic instability And accumulate, leading to the occurrence and development of malignant tumors.