【摘要】 目的:探讨自噬基因Beclin 1在人卵巢癌SKOV3细胞中过表达对其体外生长活性的影响。方法:构建自噬基因Beclin 1的真核表达载体pcDNA3.1/Beclin1,将其稳定转染SKOV3细胞,实现Beclin 1在SKOV3中的过表达;用MTT法分析Beclin1过表达对SKOV3增殖的影响,用流式细胞仪检测凋亡和自噬情况,电镜和荧光显微镜下观察自噬现象。结果:pcD-NA3.1/Beclin1转染SKOV3细胞后Beclin 1在mRNA和蛋白水平均高于空质粒pcDNA3.1转染组和未转染组。Beclin 1在SKOV3中的稳定过表达后G1期细胞明显增多,S期细胞比例明显减少,细胞增殖受到抑制,凋亡率为(21.26±3.89)%,高于空质粒pcDNA3.1转染组和未转染组,差异有统计学意义(P<0.05);流式细胞仪检测pcDNA3.1/Beclin1转染SKOV3细胞后MDC标记的自噬囊泡平均荧光强度高于pcDNA3.1转染组和未转染组。pcDNA3.1/Beclin1转染SKOV3细胞后在电镜下可见大量自噬囊泡形成。在荧光显微镜下见pcDNA3.1/Beclin1转染后SKOV3细胞中MDC标记的自噬囊泡明显增加。结论:自噬基因Beclin1过表达可诱导人卵巢癌细胞SKOV3自噬和凋亡,针对自噬基因Beclin1的卵巢癌基因治疗可能具有可行性。更多还原

【Abstract】 Objective:To explore the effect of autophagy gene Beclin 1 overexpression on the growth of human ovarian carcinoma SKOV3 cells in vitro.Methods:Eukaryotic expression vector pcDNA3.1/Beclin1 of autophagy gene Beclin 1 was constructed,then they were transfected into SKOV3 cells,Beclin1 overexpression was obtained in SKOV3 cells after transfection.MTT method was used to analyze the effect of Beclin1 overexpression on the proliferation of SKOV3 cells.Flow cytometry was used to detect apoptosis and autophagy;autophagy was observed under electron microscopy and fluorescence microscope.Results:The levels of Beclin1 mRNA and protein in SKOV-3 cells after transfected with pcDNA3.1/Beclin1 transfectant were significantly higher than those after transfected with blank plasmid and non-transfection group.After stable overexpression of Beclin1 in SKOV-3 cells,the proportion of cells at G1 phase increased significantly,the proportion of cells at S stage decreased significantly,cell proliferation was inhibited,the apoptosis rate was(21.26±3.89)%,which was significantly higher than those in blank plasmid pcDNA3.1 transfection group and non-transfection group(P<0.05);the results of flow cytometry showed that the average fluorescence intensity of autophagy vesicles labeled by MDC in SKOV-3 cells after pcDNA3.1/Beclin1 transfection was significantly higher than those in blank plasmid pcDNA3.1 transfection group and non-transfection group.A lot of autophagy vesicles were found in SKOV-3 cells after pcDNA3.1/Beclin1 transfection under electron microscopy.The number of autophagy vesicles labeled by MDC in SKOV-3 cells after pcDNA3.1/Beclin1 transfection increased significantly under fluorescence microscope.Conclusion:Autophagy gene Beclin1 overexpression can induce autophagy and apoptosis of SKOV3 cells,gene therapy targeting to autophagy gene Beclin1 may be feasible in treatment of ovarian cancer.更多还原