论文部分内容阅读
目的研究木犀草素对胃癌细胞MGC803的增殖抑制作用及其机制。方法通过MTT法、克隆形成抑制实验观察木犀草素对胃癌细胞MGC803的增殖抑制作用,碘化丙啶(PI)单染色法检测细胞周期,AnnexinⅤ-PI双染法测定细胞凋亡,蛋白质印迹法检测Bcl-2家族蛋白、Caspase蛋白、周期及自噬相关蛋白的表达情况。结果 MTT法检测发现,木犀草素作用于MGC803细胞24、48和72h后,IC50分别为127.37、76.12、16.84μmol/L;木犀草素能抑制胃癌细胞MGC803的克隆形成。PI单染色法发现随着木犀草素浓度的增大,发生G2期阻滞的细胞比例增加。蛋白质印迹法的检测结果表明,随着木犀草素浓度的增加,CDK4、CDK6、CyclinE2表达减少,CyclinD1、CyclinD3和p-Wee1表达基本不变,CyclinA、CyclinB、Myt1、p-cdc2(Tyr15)表达增高。AnnexinⅤ-PI双染法发现随木犀草素浓度增加,细胞的早期凋亡率和晚期凋亡率均增加。蛋白质印迹法的检测结果表明,Mcl-1、Bcl-xL、Caspase-3、Caspase-8、Caspase-9蛋白表达随木犀草素浓度增加而减少,Bcl-2蛋白表达量变化不大,Bax蛋白表达量增加,因而引起Bcl-2/Bax、Mcl-1/Bax和Bcl-xL/Bax的比例下降;同时可见到Caspase-3和PARP出现了切割的条带;P62表达下降,Beclin-1、LC3Ⅱ表达增加。结论木犀草素使胃癌细胞MGC803发生G2期阻滞,诱导细胞自噬的发生并抑制细胞的生长,还通过线粒体途径诱导胃癌细胞MGC803凋亡。
Objective To study the inhibitory effect of luteolin on the proliferation of gastric cancer cell line MGC803 and its mechanism. Methods MTT assay and clonogenic assay were used to observe the inhibitory effect of luteolin on the proliferation of gastric cancer cell line MGC803. The cell cycle was detected by PI staining and the apoptosis was detected by AnnexinⅤ-PI double staining. Western blotting The expression of Bcl-2 family protein, Caspase protein, cycle and autophagy related protein were detected. Results MTT assay showed that IC50 of luteolin in MGC803 cells after 24, 48 and 72 hours were 127.37, 76.12 and 16.84 μmol / L, respectively. Luteolin could inhibit the formation of MGC803 in gastric cancer cells. PI single staining found that with the increase of luteolin concentration, the occurrence of G2 phase arrest increased the proportion of cells. The results of Western blotting showed that the expression of CDK4, CDK6 and CyclinE2 decreased while the expression of CyclinD1, CyclinD3 and p-Wee1 remained unchanged and the expression of CyclinA, CyclinB, Myt1 and p-cdc2 (Tyr15) increased with the increase of luteolin concentration Increase. Annexin Ⅴ-PI double staining method showed that with the increase of luteolin concentration, the cell early apoptosis rate and late apoptosis rate were increased. The results of Western blotting showed that the expression of Mcl-1, Bcl-xL, Caspase-3, Caspase-8 and Caspase-9 decreased with the increase of luteolin concentration, while the expression of Bcl- The expression of Beclin-1, Bcl-2 / Bax, Beclin-1 / Bax and Bcl-xL / LC3 Ⅱ expression increased. Conclusion Luteolin could induce G2 arrest in gastric cancer cell line MGC803, induce cell autophagy and inhibit cell growth, and induce apoptosis of MGC803 gastric cancer cell line through mitochondrial pathway.