白花丹素对术前PF化疗杀伤舌鳞癌细胞凋亡和自噬的影响及作用机制

发布时间：2018-07-25 19:50

【摘要】：目的:研究白花丹素(Plumbagin,PB)对术前PF化疗方案杀伤舌鳞癌CAL27细胞凋亡和自噬的影响及作用机制。方法:1、人舌鳞癌CAL27细胞的细胞培养。2、MTT法分别检测PB、DDP、5-Fu对CAL27细胞的增殖抑制作用及半数抑制浓度IC50。3、MTT法检测PB联合PF方案对CAL27细胞的增殖抑制作用。4、流式细胞仪检测PB和/或PF方案作用于舌鳞癌CAL27细胞后的细胞凋亡率。5、Cyto ID荧光显微镜检测PB和/或PF方案作用于舌鳞癌CAL27细胞后的自噬水平。6、Western Blot法检测PB和/或PF方案作用于舌鳞癌CAL27细胞后自噬相关蛋白Beclin1、LC3的表达。7、Western Blot法检测PB和/或PF方案作用于舌鳞癌CAL27细胞后PI3K/AKT/mTOR信号通路蛋白的表达。结果:1、PB、DDP、5-Fu分别以浓度依赖方式抑制CAL27细胞增殖,其作用24h的半数抑制浓度(IC50)分别为7.77μmol/L、10.50μg/ml、1.26mg/ml。2、PB联合PF方案作用,与单独PF方案比较,舌鳞癌CAL27细胞的增殖抑制作用明显增强。3、PB联合PF方案作用,与单独PF方案比较,舌鳞癌CAL27细胞的凋亡率明显增强。4、PB联合PF方案作用,与单独PF方案比较,舌鳞癌CAL27细胞的自噬水平明显增强。5、当PB单独作用时,CAL27细胞中PI3K、p-AKT、p-mTOR蛋白表达不同程度下调,且PB联合PF方案比起单独应用PF方案,其细胞中PI3K、p-AKT、p-mTOR蛋白表达亦不同程度降低。6、应用PI3K/AKT激动剂IGF-1、mTOR激动剂MHY1485作用后,该激动剂逆转了PB对CAL27细胞中PI3K、p-AKT、p-mTOR蛋白的下调作用,同时显著抑制PB对PF化疗CAL27细胞凋亡和自噬诱导作用。结论:白花丹素通过抑制PI3K/AKT/mTOR信号通路诱导舌鳞癌细胞凋亡和自噬从而增强PF化疗方案杀伤舌鳞癌CAL27细胞。
[Abstract]:Aim: to study the effect and mechanism of Plumbagin PB on apoptosis and autophagy of CAL27 cells in tongue squamous cell carcinoma treated with PF regimen before operation. Methods the cell culture of human tongue squamous cell carcinoma (CAL27) cells was used to detect the proliferation inhibition of CAL27 cells by PBP 5-Fu, the inhibitory effect of PB combined with PF regimen on the proliferation of CAL27 cells by IC50.3% MTT assay, and the proliferation inhibition of PB by flow cytometry. Apoptosis rate of CAL27 cells in tongue squamous cell carcinoma treated with PF and / or PF. 5 Cyto ID fluorescence microscope to detect autophagy level of PB and / or PF on CAL27 cells of tongue squamous cell carcinoma. Western Blot assay to detect PB and / or PF regimen acting on tongue scale Expression of autophagy associated protein Beclin1hLC3 in CAL27 cells was detected by Western Blot. The expression of PI3K/AKT/mTOR signal pathway protein was detected by PB and / or PF regimen in CAL27 cells of tongue squamous cell carcinoma. Results the proliferation of CAL27 cells was inhibited in a concentration-dependent manner. The half inhibitory concentration (IC50) was 7.77 渭 mol / L (10.50 渭 g / L) and 1.26 mg / ml 路2nPB in combination with PF regimen, respectively. Compared with PF alone, the inhibitory concentration (IC50) was 7.77 渭 mol 路L ~ (-1) 路ml ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1) 路L ~ (-1), respectively. Compared with PF alone, the apoptotic rate of CAL27 cells in tongue squamous cell carcinoma was significantly increased, and compared with PF alone, the apoptotic rate of CAL27 cells in tongue squamous cell carcinoma was significantly increased. The level of autophagy in CAL27 cells of tongue squamous cell carcinoma was significantly increased. When PB was treated alone, the expression of p-mTOR protein was down-regulated in CAL27 cells, and PB combined with PF regimen was significantly lower than that of PF alone. The expression of PI3Knp-AKTor p-mTOR protein was also decreased in different degree. The agonist MHY1485, a PI3K/AKT agonist, reversed the down-regulation of PI3KPT-AKTnp-mTOR protein in CAL27 cells by using IGF-1mTOR agonist. At the same time, PB significantly inhibited apoptosis and autophagy induced by PF chemotherapeutic CAL27 cells. Conclusion: Baihuadan inhibits PI3K/AKT/mTOR signaling pathway and induces apoptosis and autophagy in tongue squamous cell carcinoma cells, thereby enhancing PF chemotherapy regimen to kill CAL27 cells.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.86

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