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目的:研究DENN-SV基因在人乳腺癌细胞系MCF-7中抗凋亡作用的可能机制。方法:将前期试验筛选出的含最佳靶点序列的菌株扩增并抽提质粒,将实验分为空白组、空白载体转染组及最佳靶点组。将实验分为两组,每组分为空白组、空白载体转染组及最佳靶点组,一组转染后48h给予TRAIL 25 ng/ml处理4 h,另一组不予TRAIL处理。收集细胞,采用流式细胞技术检测细胞凋亡率,采用Elisa法测定细胞裂解液中NF-κB含量,采用EMSA法检测核蛋白样品中NF-κB活性。结果:流式细胞仪检测TRAIL诱导凋亡效果增强,与空白组、阴性对照组相比较差异具有统计学意义(P<0.05);Elisa检测NF-κB结果示实验组的NF-κB表达量显著降低差异有统计学意义(P<0.05),EMSA检测NF-κB结果示实验组的NF-κB活性减弱差异有统计学意义(P<0.05)。结论:干扰DENN-SV基因的表达后,给予TRAIL处理可引起MCF-7细胞的凋亡率增高。这种现象的机制可能与NF-κB活性有关,且该现象与NF-κB的关系与TRAIL的作用没有直接联系。
AIM: To investigate the possible mechanism of anti-apoptotic effect of DENN-SV gene in human breast cancer cell line MCF-7. Methods: The strain containing the best target sequence screened from the previous experiment was amplified and extracted into plasmids. The experiment was divided into blank group, blank vector transfection group and the best target group. The experiment was divided into two groups, each group was divided into blank group, blank vector transfection group and the best target group. One group was treated with TRAIL 25 ng / ml for 48 hours 48 hours after transfection, the other group was not treated with TRAIL. The cells were collected and the apoptosis rate was detected by flow cytometry. The content of NF-κB in cell lysate was determined by Elisa method. The NF-κB activity in nucleoprotein samples was detected by EMSA. Results: The effect of TRAIL on apoptosis was enhanced by flow cytometry, which was significantly different from that of blank control group and negative control group (P <0.05). The results of Elisa assay showed that the expression of NF-κB in experimental group was significant The difference was statistically significant (P <0.05). EMSA detection of NF-κB results showed that the NF-κB activity in the experimental group had a significant difference (P <0.05). Conclusion: After interfering with the expression of DENN-SV gene, the apoptosis of MCF-7 cells can be induced by TRAIL treatment. The mechanism of this phenomenon may be related to NF-κB activity, and the relationship between this phenomenon and NF-κB is not directly related to the role of TRAIL.