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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。
AIM: To investigate the effect of autophagy gene Beclin 1 overexpression on the growth activity of human ovarian cancer SKOV3 cells. METHODS: The eukaryotic expression vector pcDNA3.1 / Beclin1 of autophagy gene Beclin 1 was constructed and stably transfected into SKOV3 cells to achieve the overexpression of Beclin 1 in SKOV3. The effect of Beclin1 overexpression on the proliferation of SKOV3 was analyzed by MTT assay. Apoptosis and autophagy were detected by flow cytometry. Autophagy was observed under electron microscope and fluorescence microscope. Results: The mRNA and protein levels of Beclin 1 in pcDNA3.1 / Beclin1 transfected SKOV3 cells were significantly higher than those in pcDNA3.1 transfected and untransfected cells. The expression of Beclin 1 in SKOV3 cells was significantly increased in G1 phase, significantly decreased in S phase and inhibited in cell proliferation (21.26 ± 3.89)%, which was higher than that in pcDNA3.1 transfected group (P <0.05). The average fluorescence intensity of MDC-labeled autophagy vesicles after transfected with pcDNA3.1 / Beclin1 into SKOV3 cells by flow cytometry was higher than that of pcDNA3.1-transfected and Untransfected group. After pcDNA3.1 / Beclin1 transfected SKOV3 cells, a large number of autophagic vesicles were seen under electron microscope. Under the fluorescence microscope, the number of MDC-labeled autophagic vesicles in SKOV3 cells was significantly increased after pcDNA3.1 / Beclin1 transfection. CONCLUSION: Autophagy gene Beclin1 overexpression can induce autophagy and apoptosis in human ovarian cancer cell line SKOV3, and ovarian cancer gene therapy targeting autophagy gene Beclin1 may be feasible.