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目的研究天花粉蛋白(TCS)对人宫颈癌HeLa细胞生长抑制作用及其机制,探讨HeLa细胞凋亡发生的分子机制。方法采用CCK-8的方法,检测TCS对HeLa细胞的抑制作用;流式细胞仪检测TCS对细胞周期的影响;电子显微镜和Annexin法观察TCS对HeLa细胞诱导凋亡的作用;应用Western blotting观察HeLa细胞caspase-3酶原的变化;用caspases活性检测试剂盒,检测caspase-3,8,9的活性。结果1.TCS对HeLa细胞的生长具有明显的抑制作用;2.TCS可以将HeLa细胞阻滞于S期;3.TCS诱导HeLa细胞发生凋亡,HeLa细胞出现典型的凋亡特征———染色质浓缩和凋亡小体,且凋亡率具有时间依赖性;4.caspase-3酶原表达的降低呈现时间和浓度依赖性,而caspase-3活性随时间依赖性的增高,提示在TCS诱导HeLa细胞凋亡的过程中caspase-3被激活;5.caspase-8,9的活性增高,有时间依赖性,且具有时间顺序。100 mg/L TCS处理24h后,caspase-8早于caspase-3被激活,提示活化的caspase-8可能对caspase-3起到激活作用。结论TCS对HeLa细胞的抑制作用是通过将HeLa细胞阻滞于S期和诱导细胞凋亡来实现的。caspase-3的激活是介导TCS诱导HeLa细胞发生凋亡的通路,同时,活化的caspase-8,9可能参与了caspase-3的激活和凋亡的诱导。
Objective To study the inhibitory effect and mechanism of trichosanthin (TCS) on human cervical cancer HeLa cells, and to explore the molecular mechanism of apoptosis in HeLa cells. Methods The inhibitory effect of TCS on HeLa cells was detected by CCK-8. The effect of TCS on cell cycle was detected by flow cytometry. The apoptosis of HeLa cells induced by TCS was observed by electron microscope and Annexin method. HeLa was observed by Western blotting. Changes in caspase-3 zymogens; detection of caspase-3,8,9 activity using caspases activity assay kit. Results 1. TCS has a significant inhibitory effect on the growth of HeLa cells; 2. TCS can block HeLa cells in S phase; 3. TCS induces apoptosis in HeLa cells, typical apoptosis characteristics of HeLa cells. Mass concentration and apoptotic bodies, and the apoptosis rate is time-dependent; 4. Caspase-3 proenzyme expression is reduced in a time- and concentration-dependent manner, while caspase-3 activity increases with time, suggesting that in the TCS induction Caspase-3 is activated during the apoptosis of HeLa cells. 5. The activity of caspase-8,9 is time-dependent and time-dependent. After treatment with 100 mg/L TCS for 24 h, caspase-8 was activated earlier than caspase-3, suggesting that activated caspase-8 may activate caspase-3. Conclusion The inhibitory effect of TCS on HeLa cells is achieved by blocking HeLa cells in S phase and inducing apoptosis. Activation of caspase-3 is a pathway that mediates apoptosis in HeLa cells induced by TCS. At the same time, activated caspase-8,9 may be involved in caspase-3 activation and apoptosis induction.