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目的:探讨Apoptin基因对人宫颈癌Hela细胞的抑制作用和作用方式。方法:应用脂质体转染法将重组质粒pVAX1-Apoptin和载体质粒pVAX1分别体外转染Hela细胞,作为重组质粒pVAX1-Apoptin组和载体质粒pVAX1组,同时设空白细胞对照组。应用Western blotting检测Apoptin基因在Hela细胞中的表达;运用噻唑兰(MTT)分析法检测重组质粒对Hela肿瘤细胞的抑制作用;罗丹明123和DCFA染色测定线粒体跨膜电位(ΔΨm)和活性氧水平变化;底物染色反应检测caspase-3活性。结果:pVAX1-Apoptin组转染48h后,Hela细胞的抑制率为69.28%,明显高于对照组(0.74%)和pVAX1组(10.11%)(P<0.01)。与对照组比较,pVAX1-Apoptin组线粒体ΔΨm下降(P<0.01),细胞内活性氧水平升高(P<0.05),caspase-3活性增强(P<0.01)。结论:Apoptin可通过诱导Hela细胞凋亡,进而特异性杀伤Hela肿瘤细胞。
Objective: To investigate the inhibitory effect of Apoptin gene on human cervical carcinoma Hela cells and the mode of action. Methods: The recombinant plasmid pVAX1-Apoptin and vector plasmid pVAX1 were transfected into Hela cells by liposome transfection method. The recombinant plasmids pVAX1-Apoptin and vector pVAX1 were transfected into HeLa cells with blank control group. The expression of Apoptin gene in Hela cells was detected by Western blotting. The inhibitory effect of the recombinant plasmids on Hela cells was detected by MTT assay. The mitochondrial transmembrane potential (ΔΨm) and reactive oxygen species (ROS) levels were determined by rhodamine 123 and DCFA staining Changes; Substrate staining reaction detected caspase-3 activity. Results: After 48 h of transfection, the inhibitory rate of Hela cells in pVAX1-Apoptin group was 69.28%, significantly higher than that in control group (0.74%) and pVAX1 group (10.11%) (P <0.01). Compared with the control group, mitochondrial ΔΨm of pVAX1-Apoptin group decreased (P <0.01), intracellular reactive oxygen species level increased (P <0.05) and caspase-3 activity increased (P <0.01). Conclusion: Apoptin can specifically kill Hela tumor cells by inducing Hela cell apoptosis.