响尾蛇神经毒素诱导人大细胞肺癌 NCI-H661细胞凋亡机制研究

Study on the mechanism of human large cell lung cancer NCI-H661 cells apoptosis induced by crotoxin

目的观察南美响尾蛇神经毒素（crotoxin）诱导人大细胞肺癌 NCI-H661细胞的凋亡，并初步探讨其作用机制。方法通过四唑盐还原法（CCK-8）检测 crotoxin 对 NCI-H661细胞的生长抑制率，应用细胞集落平板克隆实验观察 crotoxin 对 NCI-H661细胞集落形成的影响。实验分4组：阴性对照组、crotoxin 组（60μg/ ml crotoxin 作用24 h）、crotoxin ＋ SB203580组（5μmol/ L SB203580预处理1 h 后，60μg/ ml crotoxin 作用24 h）、SB203580组（5μmol/ L SB203580预处理1 h 后，完全培养液培养），应用流式细胞术检测 crotoxin 对 NCI-H661细胞周期及细胞凋亡的影响，并检测以 SB203580抑制 p38MAPK 活性后细胞周期及细胞凋亡率的变化。结果当 crotoxin 浓度≥30μg/ ml 时对 NCI-H661细胞生长和集落形成有抑制作用，且抑制率随着作用时间和给药浓度的增加而上升；流式细胞术检测 crotoxin 对 NCI-H661细胞周期及细胞凋亡影响结果显示 crotoxin 组、crotoxin ＋ SB203580组凋亡率分别为（16.70±1.38）％、（2.15±0.54）％，与对照组（1.47±0.29）％相比，前者差异具有统计学意义，后者差异无统计学意义（t ＝-18.763，P ＝0.000；t ＝-1.935，P ＝0.125）；crotoxin 组、crotoxin ＋ SB203580组 G1期细胞分别为（57.25±1.09）％、（48.04±1.03）％，与对照组（47.46±0.69）％相比，前者差异具有统计学意义，后者差异无统计学意义（t ＝-13.124，P ＝0.000；t ＝-0.809，P ＝0.464）。结论 crotoxin 对人大细胞肺癌NCI-H661细胞凋亡有促进作用，这一作用可能与其激发 p38MAPK信号通路活性有关。

Objective To observe the apoptosis of human large cell lung cancer NCI-H661 cells induced by crotoxin,and to explore its mechanism. Methods The growth suppression of crotoxin on the NCI-H661 cells was detected by CCK-8 colorimetry,and the formation of NCI-H661 cells was observed by the plat colony experiment. This experiment included 4 groups：negative control group,crotoxin group（60 μg/ ml crotoxin acted for 24 h）,crotoxin ＋ SB203580 group（pretreated cells using 5 μmol/ L SB203580 for 1 h,then 60 μg/ ml crotoxin acted for 24 h）,SB203580 group（pretreated cells using 5 μmol/ L SB203580 for 1 h,then cultivated cells using complete culture solution）. They were detected that the cell cycle and apoptosis rate of NCI-H661 cells treated with crotoxin by the flow cytometry. Additionally,they were tested that the change of the cell cycle and apoptosis rate after the NCI-H661 cells were treated with crotoxin and the activity of p38MAPK was inhibited by SB203580. Results When the concentration of crotoxin was greater than or equal to 30 μg/ ml,the inhibitory effect of crotoxin on the activity of NCI-H661 cells and colony formation,and inhibition rate rose with increasing function of time and drug concentration. Flow cytometry showed that the apoptosis rate of crotoxin group and crotoxin ＋ SB203580 group were（16. 70 ± 1. 38）% and（2. 15 ± 0. 54）% ,com-pared to the control group（1. 47 ± 0. 29）% ,and the former difference was statistically significant and the latter was not statistically significant（t = - 18. 763,P = 0. 000;t = - 1. 935,P = 0. 125）. The G1 period cells of nbsp;crotoxin group and crotoxin ＋ SB203580 group were（57. 25 ± 1. 09）% and（48. 04 ± 1. 03）% ,compared to the control group（47. 46 ± 0. 69）% ,and the former difference was statistically significant and the latter was not statistically significant（t = - 13. 124,P = 0. 000;t = - 0. 809,P = 0. 464）. Conclusion Crotoxin can promote the apoptosis of human large cell lung cancer NCI-H661 cells,and this effect may be related to the excitation of p38^MAPK signal pathway.