摘要
目的探讨三叶青提取物对人肺癌H1299细胞增殖抑制、凋亡作用及其机制。方法采用Cell Counting Kit-8(CCK-8)检测三叶青提取物对H1299细胞增殖的抑制作用;采用倒置显微镜观察三叶青提取物对H1299细胞形态的影响;荧光显微镜观察三叶青提取物作用细胞后经Hoechst 33258染色的凋亡形态变化;流式细胞仪检测细胞凋亡率;蛋白印迹法(Western blot)检测pro-caspase-3、9,cle-caspase-3、9,poly-ADP-ribose polymerase(PARP)蛋白表达变化。结果与对照组比较,浓度为0.5、1、5、10 mg/m L三叶青提取物作用H1299细胞后抑制率升高(P<0.05,P<0.01);同时作用时间越长,抑制率越高(P<0.01)。倒置显微镜下观察给药后细胞形态明显改变,细胞减少;荧光显微镜下观察荧光染色后细胞核浓染和凋亡小体形成。流式细胞仪检测结果显示细胞凋亡率呈明显的量-效关系。Western blot结果表明:与对照组比较,浓度为5、10 mg/m L三叶青提取物明显抑制H1299细胞中pro-caspase-3、9和PARP蛋白表达,增加cle-caspase-3、9,cle-PARP蛋白的表达,差异有统计学意义(P<0.05)。结论三叶青提取物对人肺癌H1299细胞具有明显抑制其增殖,促进细胞凋亡作用,且其机制可能与激活caspase蛋白表达有关。
Objective To study the effect of extract of Radix Tetrastigma hemsleyani on the proliferation and apoptosis of human lung carcinoma H1299 cells,and to explore its mechanisms. Methods H1299 cells were treated with the extract of Radix Tetrastigma hemsleyani in different concentrations at different time points. Its inhibition on H1299 cell proliferation was detected by Cell Counting Kit-8（ CCK-8） assay. The morphology of the H1299 cell was observed by inverted microscope. Changes of apoptosis were observed by Hoechst33258 methods. The apoptosis rate was detected by flow cytometry. Expression changes of apoptosis-related proteins pro-caspase-3,pro-caspase-9,cle-caspase-3,cle-caspase-9,and poly-ADP-ribose polymerase（ PARP） were detected by Western blot. Results Compared with the control group,the inhibition rate of H1299 cells increased after acted by 0. 5,1,5,and 10 mg / m L extract of Radix Tetrastigma hemsleyani（ P 〈0. 05,P 〈0. 01）. The longer the acting time,the higher the inhibition rate（ P 〈0. 01）. Under inverted microscope,typical morphological changes could be seen and the number of H1299 cells was reduced. Under fluorescence microscope,dark stained nucleus and formed apoptotic body could be observed. Results of flow cytometry showed that the apoptosis rate was obviously dose-effect correlated with the concentration of extract of Radix Tetrastigma hemsleyani. Results of Western blot indicated that compared with the control group,the protein expression of pro-caspase-3,pro-caspase-9,and PARP were down-regulated and that of cle-caspase-3,cle-caspase-9,and cle-PARP were up-regulated by 5 and 10 mg / m L extract of Radix Tetrastigma hemsleyani（ P 〈0. 05）. Conclusions Extract of Radix Tetrastigma hemsleyani had obvious effect in inhibiting the proliferation and inducing apoptosis of human lung carcinoma H1299 cells,which might be achieved by activating the expression of caspase protein.
出处
《中国中西医结合杂志》
CAS
CSCD
北大核心
2014年第11期1354-1358,共5页
Chinese Journal of Integrated Traditional and Western Medicine
基金
浙江省科技计划项目(No.2008F3036)
浙江省科技厅科研院所专项基金资助项目(No.011F10068)
关键词
三叶青
人肺癌细胞
增殖
凋亡
Radix Tetrastigma hemsleyani
human lung carcinoma cell
proliferation
apoptosis
