冬凌草甲素诱导胃癌细胞MKN45凋亡的实验研究

目的探讨冬凌草甲素对胃癌细胞MKN45生长及调亡的影响及其机制。方法MTT法检测冬凌草甲素对MKN45细胞生长抑制作用：AO／EB和Hoechest33258荧光染色法及倒置显微镜观察细胞形态学变化：单细胞分析系统PI单染检测细胞周期变化：采用单细胞分析系统Annexinv—PE和7-AAD双染色法检测细胞凋亡率：Western blot检测凋亡相关蛋白Bel-2、Bax和caspase-3的表达改变。结果冬凌草甲素对胃癌细胞MKN45具有明显的生长抑制作用，普通形态学、AO／EB及Hoechest33258荧光染色均可见典型的细胞凋亡改变。冬凌草甲素作用MKN45细胞12h后的细胞凋亡率为8．7％～17．9％，24h后的细胞凋亡率为13．9％．29．3％，与未加药对照组（12h：3．3％；24h：4．8％）比较，差异具有统计学意义（P〈0．01）。细胞周期分析显示冬凌草甲素使MKN45细胞增殖阻滞于G2-M期。Western blot检测显示．冬凌草甲素作用MKN45细胞后，Bax和caspase-3蛋白的表达随着药物作用浓度增高而增加．而Bcl-2蛋白表达无明显变化．Bcl-2／Bax比值减低。结论冬凌草甲素对胃癌细胞MKN45具有明显的增殖抑制作用：冬凌草甲素可能通过增加Bax蛋白表达．改变Bcl-2／Bax比率．继而启动caspase途径诱导胃癌细胞MKN45凋亡。

Empirical study of oridonin-induced gastric cancer cells MKN45 apoptosis

Objective To investigate the growth inhibition and apoptosis of gastric cancer cell MKN45 induced by oridonin and its mechanism. Methods The MTT method was used to investigate the inhibitory effect of oridonin on MKN45 cells. The AO/EB and Hoechest33258 staining were used to observe the cell morphologic changes of apoptosis induced by oridonin. Prophase apoptotic ratio and cell cycle change were evaluated by GuavaEasycyte PCA-96 system. The expressions of Bcl-2, Bax and caspase 3 proteins were determined by Western blot. Results Oridonin significantly inhibited the proliferation of MKN45 cells in dose- and time-dependent manner. Typical apoptotic features of the cells treated with oridonin were found by AO/EB and Hoechest33258 staining. When MKN45 cells were treated with different doses of oridonin for 12 h, the prnphase apoptotic ratio was stepped up from 3.3% (untreated group) to 8.7%-17.9%; after 24 h, from 4.8%(untreated group) to 13.9%-29.3%. There was significant difference between treated and untreated groups (P<0.01). After treatment with oridonin for 24 h, MKN45 cells were arrested at G<,2/M phase. Western blot analysis showed up-regulated expression of Bax and caspase-3, and no significant change of Bcl-2, but Bcl-2/Bax ratio decreased significantly. Conclusions Oridonin significantly inhibits the proliferation of MKN45 cell. Apoptosis of MKN45 induced by oridonin may be associated with the up-regulated expression of Bax and the change of Bcl-2/Bax ratio, thus to activate the caspase pathway.