基于微流控芯片技术橙皮苷抗肺肿瘤作用机制研究

摘要

目的 探究橙皮苷诱导人肺癌细胞A549凋亡的作用机制.方法 基于微流控芯片技术采用Hoechst 33342/PI染色法检测橙皮苷对肺癌细胞A549凋亡坏死的影响;流式细胞术检测橙皮苷对肺癌细胞周期及凋亡的影响;实时荧光定量PCR技术检测相关基因VEGF、PI3K及PTEN的表达;Western blot 技术检测橙皮苷诱导肺癌细胞中PI3K-Akt信号通路相关蛋白PI3K、Akt、PTEN及凋亡蛋白Bcl-2、周期蛋白Cyclin B1的表达. 结果 橙皮苷作用于细胞G0/G1期及S期,阻滞细胞分裂,并呈现浓度依赖性诱导肺癌细胞凋亡,其细胞凋亡坏死率由正常对照组的(6.7±0.6)%增至(27.9±1.1)%;经橙皮苷诱导后的肺癌细胞中VEGF和PI3K基因表达相对降低,抑癌基因PTEN表达相对增加.Western blot 结果显示,经橙皮苷诱导的肺癌细胞中凋亡蛋白Bcl-2及周期蛋白Cyclin B1相对表达降低,PI3K-Akt信号通路蛋白Akt表达量与对照组比较明显减少,PI3K蛋白表达量相对增加,PTEN表达量无明显变化.结论 橙皮苷可能是通过干扰PI3K-Akt信号通路,阻碍肿瘤细胞的分裂并促进凋亡蛋白的产生,从而诱导肺癌细胞 A549 凋亡.%Aim To investigate the effect of hesperidin on human lung cancer cell A549 and the possible mechanism.Methods The cell apoptosis and necrosis of A549 treated with hesperidin were measured by the Hoechst 33342/PI fluorescent dye based on microfluidic chip technology.Cell cycle and apoptosis rate were evaluated by flow cytometry(FCM).The expressions of the related genes were detected through the real-time fluorescent quantitative PCR technology(RT-PCR) including VEGF, PI3K and PTEN.The protein expressions of Bcl-2, Cyclin B1, PI3K, Akt and PTEN were detected by Western blot after hesperidin intervention.Results The proliferation of A549 cells was significantly inhibited by hesperidin in a dose-dependent manner.FCM results showed that hesperidin could not only influence the G0/G1 phase and S phase, but also promote the apoptosis of lung cancer cells.Meanwhile, the apoptosis and necrosis rate was increased from(6.7±0.6)% to(27.9±1.1)% compared with that of control group(P<0.05).From the level of molecular, the gene expressions of VEGF and PI3K were decreased, while the PTEN was increased after hesperidin stimulation.Western blot results showed that the expression of protein Bcl-2, Cyclin B1 and Akt were decreased, which all showed close relationship with cell apoptosis, cell cycle and PI3K-Akt signaling pathway.The expression of PI3K was increased, but the change of PTEN was not statistically significant compared with that of control group.Conclusion Hesperidin induces lung cancer cell apoptosis through PI3K-Akt signaling pathway, which blocks cancer cell division and destroys the balance of related protein expression.