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目的研究肿瘤特异性凋亡蛋白基因(apoptin)在诱导人宫颈癌细胞凋亡过程中信号转导机制。方法用含有apoptin基因的真核表达载体瞬间转染体外培养的人宫颈癌细胞(hela);采用逆转录-聚合酶链反应(RT-PCR)、DNA凝胶电泳、流式细胞术检测hela细胞的凋亡;以比色法检测胱天蛋白酶-8(caspase-8)和胱天蛋白酶-3(caspase-3)的相对活性。结果 Apoptin基因瞬间转染的hela细胞可出现典型的细胞凋亡所具有的DNA梯状带;流式细胞术检测发现,实验组细胞凋亡率〔(40.45±0.76)%〕明显高于正常对照组〔(3.25±0.16)%〕和载体对照组〔(3.55±0.12)%〕(P<0.01);caspase-3的活性升高,但caspase-8活性无明显变化。结论 Apoptin基因可通过激活caspase-3诱导hela细胞凋亡。
Objective To study the signal transduction mechanism of apoptin in inducing human cervical cancer cell apoptosis. Methods Hela cells were transiently transfected with eukaryotic expression vector containing apoptin gene. Hela cells were detected by reverse transcription polymerase chain reaction (RT-PCR), DNA gel electrophoresis and flow cytometry . The relative activities of caspase-8 and caspase-3 were detected by colorimetric assay. Results Hela cells transiently transfected with Apoptin gene showed typical ladder DNA ladder. The results of flow cytometry showed that the apoptotic rate in the experimental group 〔(40.45 ± 0.76)%〕 was significantly higher than that in the normal control (3.25 ± 0.16)%] and vehicle control group (3.55 ± 0.12%) (P <0.01). The activity of caspase-3 increased but the activity of caspase-8 did not change significantly. Conclusion Apoptin can induce hela cell apoptosis by activating caspase-3.