摘要
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 The purpose of the current study is to investigate the growth inhibition and the apoptosis of HeLa cells induced with TCS, and to explore the underlying molecular mechanism. Methods The antiproliferative effect of TCS on HeLa cells was measured with CCK-8. The FCM analysis was carried out to examine the effect of TCS on the cycle distribution of HeLa cells. Electron microscopy and FCM analysis were employed to observe the apoptosis of HeLa cells. The protein expression of caspase-3 was determined by Western blotting. The activity of caspase-3, 8, 9 was determined by a caspases colorimetric assay kit. Results 1.The proliferation of HeLa cells was significantly inhibited by TCS; 2.TCS led a S-phase arrest in cell cycle; 3.HeLa cell apoptosis was induced with TCS, which was characterized by chromatin condensation and apoptotic bodies. And the apoptotic rate of HeLa cells increased with time; 4.The expression of caspase-3 protein was decreased significantly （P 〈 0.01） in a dose- and time-dependent manner and the activity of caspase-3 was increased with a time-dependent, which clearly indicated that caspase-3 was activated in the apoptosis of HeLa cells; 5.The activity of caspase-8, 9 also had a time-dependent increase in the experiment. In terms of temporal sequence of the caspase activation, caspase-8 was the first caspase triggered at 24h following 100mg/L TCS treatment, which was earlier than the activation of caspase-3. These results indicated that the activation of caspase-8 led to caspase-3 activated. Conclusion The results of our study showed that TCS inhibits the growth of HeLa cell through cell cycle arrestted in S phase and the apoptosis of HeLa cells, and the studies demonstrated also that the activation of caspase-3 is a pathway mediating the apoptosis of HeLa cells induced with TCS, in which caspase-8, 9 are critically involved.
出处
《解剖学报》
CAS
CSCD
北大核心
2006年第3期309-314,共6页
Acta Anatomica Sinica
基金
浙江省科技厅资助项目(2003C30057)
浙江省分析测试基金项目(04143)
