蟾蜍灵诱导肝癌Huh7细胞的凋亡及其机制研究

The study of liver cancer huh7 cell apoptosis induced by bufalin and its mechanism

目的:研究蟾蜍灵(bufalin)诱导肝癌Huh7细胞的凋亡及其凋亡的初步机制。方法:CCK-8法检测不同浓度蟾蜍灵作用细胞不同时间后的细胞活性;Annexin V-FITC/PI双染试剂盒检测蟾蜍灵诱导的细胞死亡方式并分析细胞凋亡率;细胞活性氧ROS特异性抑制剂NAC预处理细胞1小时后,检测蟾蜍灵对细胞活性的影响,并采用Hoechst 33342染色技术染细胞核,在共聚焦显微镜下观察NAC对细胞凋亡的影响;采用流式细胞仪检测NAC对蟾蜍灵引起的细胞线粒体膜电位下降的影响。结果:10^(-9)、10^(-8)、10^(-7)、10^(-6)、10^(-5)mol/L蟾蜍灵分别作用细胞12、24、48小时后,细胞的活性呈浓度和时间依赖性降低;用10^(-7)mol/L这一浓度为接下来的实验浓度,流式双染结果表明10^(-7)mol/L蟾蜍灵诱导的细胞死亡为凋亡,凋亡率为42.9%,与星形孢菌素组引起的细胞凋亡分布类似;NAC预处理细胞1小时,再用蟾蜍灵作用细胞24小时后细胞活性为60.9±2.5%,明显比没有NAC预处理的细胞活性高,而且共聚焦显微镜和流式细胞核染色结果分别显示NAC预处理细胞可以减少蟾蜍灵引起的细胞凋亡和升高蟾蜍灵引起的线粒体膜电位的下降,升高的百分率为9.5%。结论:蟾蜍灵可以诱导肝癌细胞Huh7凋亡,ROS参与了蟾蜍灵诱导的细胞凋亡和线粒体膜电位的下降,本实验结果将为蟾蜍灵的临床使用和研究提供实验依据。

Objective： Studying the apoptosis of liver cancer Huh7 cell induced by Chinese herbs extractive bufalin and its mechanism. Methods： CCK-8 method detected the cell viability of Huh7 cell treated by different concentration of bufalin for various time ; Annexin V-FITC/PI stai- ning method was used to detect the cell death type induced by bufalin and the apoptosis ratio was calculated by flow cytometry. After pretrea- ting with NAC, reactive oxygen species （ROS） special inhibitor, for 1 hour, the cell viability induced by bufalin was detected, and further the cell was stained by Hoechst 33342 dye that was then observed under confocal microscopy; Next, flow cytometry was used to detect the effect of NAC on the mitochondrial membrane potential decline induced by bufalin. Results： 10.9, 10-4, 10-7, 104, 10Smol/L of bufalin treating cells for 12, 24 and 48 hour respectively showed concentration- and time-dependent cell viability decline. The concentration of 10-2 mol/L was selected to use in the next experiments. The cell death induced by bufalin was apoptosis, and its apoptosis ratio was 42.9%, which was similar with the distribution of positive control group. After pretreating with NAC for 1 hour, bufalin induced 60.9 ＋2.5% of cell viability which was significantly higher than that non-pretreating cells （P 〈 0.05 ）. Moreover, the results of confoeal microscopy and nucleus staining respective showed pretreating with NAC could decrease bufalin induced apoptosis and increase the bufalin induced mitochondrial membrane potential that was about 9.5 % higher （ P 〈 0.05 ）. Condusions： Bufalin could induce Huh7 cell apoptosis, and ROS participated the bufalin induced apoptosis and decline of mitoehondrial membrane potential. The results of here would provide a theoretical foundation for the clinic application and researches of bufalin.