健脾解毒方对胃癌细胞凋亡及基因调控机制的研究

Study on the Mechanism of Jianpi Jiedu Prescription Inducing Apoptosis and Gene Regulation of Gastric Cancer Cells

目的:研究健脾解毒方对胃癌细胞凋亡及基因调控机制。方法:96孔板接种对数生长期SGC-7901细胞,接种浓度是5×104个/mL,以10、20、40μg/mL健脾解毒方分别作用于胃癌SGC-7901细胞24 h。倒置显微镜下察细胞形态;MTT法检测细胞活力;Annexin V-FITC/PI双染法检测细胞凋亡率;JC-1染色检测线粒体膜电位;运用Real-time PCR检测B淋巴细胞瘤-2相关X蛋白(Bax)、细胞色素C (Cytochrome c)、自杀相关因子(Fas)、半胱氨酸蛋白酶-8 (Caspase-8)基因表达;Westernblot检测Bax、Cytochrome c、Fas、Caspase-8蛋白表达。结果:与对照组比较,20、40μg/mL健脾解毒方组细胞抑制率较高,差异均有统计学意义(P <0.05),并且呈剂量依赖性。与对照组比较,10μg/mL的健脾解毒方处理24 h后,SGC-7901细胞形态学未有明显改变;20μg/mL的健脾解毒方处理24 h后,SGC-7901细胞形态学发生改变,细胞变圆;40μg/mL处理后的细胞皱缩,数目明显的减少。10、20、40μg/mL健脾解毒方处理SGC-7901细胞后,分别有5.98%、14.94%和31.88%细胞出现凋亡。与对照组比较,10、20、40μg/mL健脾解毒方组细胞凋亡率显著升高(P <0.05),并呈浓度依赖性。随着健脾解毒方浓度的增加,SGC-7901细胞的线粒体膜电位呈浓度依赖性下降(P <0.05)。与对照组比较,10、20、40μg/mL健脾解毒方组处理SGC-7901细胞24 h后,SGC-7901细胞中的Bax、Cytochrome c、Fas、Caspase-8的基因表达明显升高(P <0.05,P <0.01),并呈剂量依赖性。20、40μg/mL健脾解毒方处理SGC-7901细胞24 h后,随着药物浓度的增加,Bax、Cytochrome c、Fas、Caspase-8蛋白表达含量也增加(P <0.05,P <0.01),且呈浓度依赖性。结论:健脾解毒方能够剂量依赖性的诱导细胞发生凋亡可能与影响细胞线粒体膜电位和Bax、Cytochrome c、Fas、Caspase-8蛋白的表达相关。

Objective： To study the mechanism of Jianpi Jiedu prescription for cell apoptosis of gastric cancer cells and gene regulation. Methods： Inoculated a 96-well plate with SGC-7901 cells in logarithmic growth phase at a concentration of 5 × 10^4 cells/mL. The gastric cancer SGC-7901 ceils were respectively treated with 10, 20, 40 μg/mL of Jianpi Jiedu prescription for 24 h. Observed the cell morphology under the inverted microscope; detected cell viability by MTT method： detected cerr apoptosis rate by Annexin V-FITC/PI double staining; detected mitochondrial membrane potential by JC-1 staining; detected gene expression of B lymphocyte tumor-2 associated X protein（Bax）, cytochrome C （Cytochrome c）, Factor associcated suicide （Fas） and caspase-8 （Caspase-8） by Real-time PCR; detected proten expression of Bax, Cytochrome c, Fas and Caspase-8 by Western-blot. Results： Compared with the control group, the inhibitory rates of cells treated with 20 and 40 μg/mL of Jianpi Jiedu prescription were higher and showed a dose-dependent manner, differences being significant （P 〈 0.05）. Compared with the control group, the cytomorphology of SGC-7901 cells did not change significantly after being treated by 10 μg/mL of Jianpi Jiedu prescription for 24 h; the cytomorphology of SGC-7901 cells changed and turned round after being treated by 20 μg/mL of Jianpi Jiedu prescription for 24 h; cells treated with 40 μg/mL of Jianpi Jiedu prescription shrunk and the number was significantly reduced 5.98% , 14.94% and 31.88% of the cells respectively showed apoptosis after SGC-7901 cells were treated by 10, 20, 40 μg/mL of Jianpi Jiedu prescription. Compared with the control group, the apoptosis rates of cells in the groups of 10, 20 and 40 μg/mL of Jianpi Jiedu prescription were significantiy increased （P 〈 0.05）, and they were concentration-dependent. With the increase of the concentration of Jianpi Jiedu prescription, the mitochondrial membrane potential of SGC-7901 cells were decreased in a concentration-dependent manner（P 〈 0.05）. Compared with the control group, the gene expressions of Bax, Cytochrome c, Fas and Caspase-8 in SGC-7901 cells were significantly increased after the cells were treated by 10, 20, 40 μg/mL of Jianpi Jiedu prescription for 24 h（P 〈 0.05, P 〈 0.01）, and they showed a dose dependent manner. After the cells were treated by 20 and 40 μg/mL of Jianpi Jiedu prescription for 24 h, the content of expressions of Bax, Cytochrome c, Fas and Caspase-8 proteins was increased with the increase of drug concentration （P 〈 0.05, P 〈 0.01）, and showed a concentration-dependent manner. Conclusion. The mechanism of Jianpi Jiedu prescription inducing cell apoptosis in a dose-dependent manner may be related to its influence on mitochondrial membrane potential and the expressions of Bax, Cvtochrome c, Fas and Caspase-8 proteins.