平肺浸膏对人肺腺癌A549细胞的杀伤作用

A549 Growth Inhibition and Apoptosis Induced by Liquid Extract of Pingfei Prescription

目的:研究中药复方平肺浸膏对人肺腺癌A549细胞增殖的影响及诱导细胞凋亡的作用。方法:采用CCK-8比色法和磷脂酰丝氨酸外翻分析法分别检测平肺浸膏对人肺腺癌A549细胞增殖和细胞凋亡的作用。平肺浸膏(1 g.mL-1)由中日友好医院药学部提供,实验前经定性滤纸粗滤,0.22μm滤膜过滤后分装。人肺腺癌A549细胞常规复苏、培养及传代,处于对数生长期开始用于实验。实验分为空白对照、生药终浓度1,5,10 g.L-1共4组。细胞以7×104/mL接种于96孔培养板,24 h细胞贴壁后加不同浓度平肺浸膏,对照组只换液不加药。分别于加药后24,48,72 h用CCK-8法检测细胞增殖情况。将细胞以3×104/mL接种于6孔培养板,5 h细胞贴壁后加不同浓度的平肺浸膏。药物作用17 h后收获细胞,应用磷脂酰丝氨酸外翻分析法,于流式细胞仪检测细胞凋亡。结果:平肺浸膏生药5,10 g.L-1在体外作用24 h以上表现出对A549细胞增殖的抑制作用,与常规对照组相比存在统计学差异(P<0.05),且这种抑制作用随时间和药物浓度增加而增强。生药10 g.L-1可诱导早期细胞凋亡,与常规对照组相比存在统计学意义(P<0.05)。结论:中药复方平肺浸膏能够抑制人肺腺癌细胞A549的增殖,诱导细胞凋亡。

Objective： To investigate the growth effect on human lung adenocarcinoma cell line A549 by liquid extract of Pingfei prescription, a traditional Chinese medicine （TCM） formula that has been used to treat lung cancer. Method： Liquid extract of Pingfei prescription （1 g· mL-1） was provided by pharmaceutical department in China-Japan friendship hospital. The liquid extract was filtrated through primary filtration by qualitative filter paper and second filtration by 0.22 Ixm microporous membrane before applied to experiments. A549 cells were recovered, cultured and subcultured. Cells in logarithmic growth phase were prepared for single cell suspension in the following experiments. Groups were blank control, liquid extract of Pingfei prescription 1, 5, 10 g -L-1 group. A549 ceils were seeded in 96-well plates, 7 ×104 per well, 3 wells for replica. Drugs were then added 24 hours after except for the blank control group, which was replaced with fresh medium only. A549 growth was detected by Cell Counting Kit-8 （CCK-8, Donjino, Japan） at 24, 48, 72 h after drugs being added. For apoptosis study, A549 cells were seeded in 6-well plates with 3× 104 per well, 3 for replica. Drugs were then added 5 hours after except for the blank control group, which was replaced with fresh medium only. Ceils were harvested 17 hours after drugs added. Apoptosis was detected by analyzing plasma membrane translocation of phosphatidylserine （PS） （FITC Annexin V Apoptosis Detection Kit i , BD Pharmingen）, using FACS. Each experiment was repeated at least 3 times. Statistic analysis for quantitative data is student t-test. Result： 5, 10 g · L-1 groups presented inhibitive effect on A549 growth at 24, 48, 72 h time points （P 〈 0.01 ）. 10 g ·L-1 group showed early and late apoptosis induction （P 〈 0.01 ）. Conclusion： Liquid extract of Pingfei prescription, a TCM formula, could suppress A549 growth in vitro in dose and time dependent manner. This inhibitory effect may be related to its apoptosis induction.