毒毛旋花子阿洛糖苷诱导胃腺癌细胞 SGC -7901凋亡作用及机制研究

摘要

Objectjve To explore the apoptosis - inducing effect of Strophalloside on gastric adenocarcinoma cells type SGC - 7901 and its mechanism. Methods The human gastric adenocarcinoma cells type SGC - 7901 were divided into control group,0. 125 μg/ ml group,0. 250 μg/ ml group,0. 500 μg/ ml group,and 1. 000 μg/ ml group. The growth inhibition effect of Strophalloside on gastric adenocarcinoma cells type SGC - 7901 was detected by MTT assay,and the apoptosis was measured by Annexin V - FITC/ PI flow cytometry and Hoechst staining. The changes of mitochondrial membrane potential were examined by JC - 1 kit. The expressions of mitochondrial pathway - related protein cytochrome C,Caspase - 3 and Caspase - 9 were detected by Western blotting analysis. Results According to MTT assay results,there were significant differences in value A and rate of growth inhibition of gastric adenocarcinoma cells type SGC - 7901 among different groups at 24 h and 48 h after treatment, respectively(P < 0. 05);comparison of the above two indicators between groups showed that value A of gastric adenocarcinoma cells type SGC - 7901 decreased sequentially,and rate of growth inhibition increased sequentially(P < 0. 05). By fluorescence microscope,the cell nucleus of control group showed homogeneous blue fluorescence,the cell nucleus of 0. 500 μg/ ml group showed compact bright fluorescence with white color. There was significant difference in total relative mortality of gastric adenocarcinoma cells type SGC - 7901 among different groups( P < 0. 05);comparison of the above indicator between groups showed that total relative mortality of gastric adenocarcinoma cells type SGC - 7901 increased sequentially( P < 0. 05 ). According to results of JC - 1 staining,gastric adenocarcinoma cells type SGC - 7901 showed green fluorescence 12 h after Strophalloside treatment,and fluorescence intensity increased as concentration of Strophalloside rised. According to Western blotting results,cytochrome C was released from mitochondria into cytoplasm C,and Caspase - 3 and Caspase - 9 were activated,shear and activation occurred,the expression level of activated fragments increased. Conclusjon Strophalloside can induce apoptosis of gastric adenocarcinoma cells type SGC - 7901 through the mitochondria - mediated pathway.%目的：探讨毒毛旋花子阿洛糖苷诱导胃腺癌细胞 SGC -7901凋亡作用及其机制。方法将人胃腺癌细胞 SGC -7901分为对照组、0.125μg/ ml 组、0.250μg/ ml 组、0.500μg/ ml 组、1.000μg/ ml 组，四甲基偶氮唑盐微量酶反应比色法（MTT）检测毒毛旋花子阿洛糖苷对胃腺癌细胞 SGC -7901的生长抑制作用，Hoechst 染色和 Annexin V - FITC/碘化丙啶（PI）流式细胞术检测细胞凋亡情况，JC -1法检测线粒体膜电位变化，Western blotting 法检测线粒体途径相关蛋白细胞色素 C、Caspase -3和 Caspase -9的表达。结果 MTT 结果显示，24 h 和48 h 时不同组胃腺癌细胞 SGC -7901吸光度（A）、生长抑制率比较，差异均有统计学意义（P <0.05）；组间两两比较胃腺癌细胞 SGC -7901 A 值依次降低、生长抑制率依次升高（P <0.05）。荧光显微镜下观察发现，对照组细胞核显示为蓝荧光且较均匀，0.500μg/ ml组处理12 h 后细胞核呈紧密较亮荧光体，颜色较白。不同组胃腺癌细胞 SGC -7901总相对死亡率比较，差异有统计学意义（P <0.05）；组间两两比较胃腺癌细胞 SGC -7901总相对死亡率依次升高（P <0.05）。JC -1染色结果显示，毒毛旋花子阿洛糖苷处理胃腺癌细胞 SGC -790112 h 后，0.125μg/ ml 组、0.250μg/ ml 组、0.500μg/ml 组显示绿色荧光，随毒毛旋花子阿洛糖苷浓度的增加绿色荧光增强。Western blotting 法显示，随着细胞凋亡的发生，细胞色素 C 从线粒体中释放到细胞质中，同时，Caspase -3和 Caspase -9也被激活并发生剪切活化，活化片段表达增加。结论毒毛旋花子阿洛糖苷通过线粒体凋亡途径诱导体外培养的人胃腺癌细胞 SGC -7901凋亡。