【作者】 李蓉；

【导师】 谭小武；

【作者基本信息】 南华大学 ， 内科学（专业学位）， 2015， 硕士

【摘要】 目的:以人肺腺癌A549细胞为研究对象,探讨黄芪多糖与顺铂对该细胞的增殖抑制、周期调控、诱导凋亡的影响,并对两药联合作用的机制进行了初步的研究,从而为非小细胞肺癌的化疗提供新的思路,为以后临床应用提供理论依据。方法:体外培养人肺腺癌A549细胞,取处于对数生长期的细胞进行实验,实验分四组:阴性对照组;黄芪多糖组;顺铂组;黄芪多糖+顺铂组。MTT法检测黄芪多糖、顺铂及两药联合对细胞的增殖影响,并用Q值公式评估两药联合的作用效果;流式细胞术检测药物对细胞周期及凋亡率的影响;RT-PCR技术检测凋亡相关蛋白Bcl-2、Bax、Caspase-3的m RNA表达水平;Western blot检测Bax、Bcl-2、Caspase-3的蛋白表达变化。结果:1、黄芪多糖、顺铂对人肺腺癌A549细胞增殖呈时间-浓度依赖抑制作用;联合用药的增殖抑制作用优于单一用药,两药联合作用表现为单纯相加。2、流式细胞术检测处理48h的A549细胞,黄芪多糖作用后可出现G1期细胞阻滞(P<0.01);顺铂作用后出现S期细胞阻滞(P<0.01);两药物联用将细胞阻滞于G1和S期(P<0.01)。黄芪多糖、顺铂均能诱导A549细胞凋亡,且同一作用时间两者联合的凋亡率高于单一用药(P<0.01)。3、黄芪多糖、顺铂均可促进人肺癌A549细胞Bax、Caspase-3 m RNA表达和抑制Bcl-2 m RNA表达,差异均有统计学意义(P<0.01)。与单一用药组比较,黄芪多糖及顺铂联合作用效果更强,差异均有统计学意义(P<0.01)。4、与阴性对照组比较,黄芪多糖、顺铂均可明显促进人肺癌A549细胞Bax、Caspase-3蛋白表达,而抑制Bcl-2蛋白表达;与单一药物组及阴性对照组比较,黄芪多糖与顺铂联合作用效果更强,差异均有统计学意义(P<0.01)。结论:黄芪多糖、顺铂具有抑制人肺腺癌A549细胞增殖及促进细胞凋亡的作用,两药联合效果明显增强。黄芪多糖协同顺铂促人肺腺癌A549细胞凋亡,其作用机制可能与下调Bcl-2表达、上调Bax表达、增强Caspase-3表达有关。更多还原

【Abstract】 Objectives In order to observe the inhibitory effects of two drugs combined astragalus polysaccharides and cisplatin on the lung adenocarcinoma A549 cells’ proliferation, cycle regulation, apoptosis induction and synergistic mechanism of the two drugs, which provided new methods and established theoretical basis for the future clinical application on non-small cell lung cancer chemotherapy.Methods Lung adenocarcinoma A549 cells were cultured in vitro,then selected the cells in logarithmic growth phase experiments were divided into four groups: the negative control group,the astragalus polysaccharide group,the cisplatin group, the Astragalus polysaccharide and cisplatin groups.MTT assay was performed to detect the effects of astragalus polysaccharides, cisplatin and the two combination on cell proliferation, then the joint effects of two medicines evaluated by the Q-value formula. The impact of each group of cell cycle and apoptosis rate are analyzed by flow cytometry. The mRNA expression of apoptosis-related protein Bcl-2, Bax and Caspase-3 levels was detected by RT-PCR.Western blot was used to detect the protein expression of Bax, Bcl-2 and Caspase-3.Results 1.Astragalus polysaccharides, or cisplatin and or the doublet could inhibit lung adenocarcinoma A549 cells’ proliferation on concentration and time-dependent manner. The combination on inhibition was superior to single medicine, but the synergistic and additive antitumor effect of the combination on the A549 cells.2. After treated the A549 cells with different groups for 48 h, the results showed that astragalus polysaccharides and cisplatin could respectively arrest cell cycles in G1 and S phase(P<0.01), while the combination could arrest cell cycles in G1, S phases(P<0.01). Astragalus polysaccharides and cisplatin could induce apoptosis of the A549 cells and the apoptosis rate of the combination was superior to single medicine(P<0.01). 3. Astragalus polysaccharides and cisplatin can promote expression of Bax and Caspase-3 mRNA, while the expression of Bcl-2 mRNA were statistically significant(P<0.01). Compared to single drug group, astragalus polysaccharides and Cisplatin effect was stronger, the differences were statistically significant(P<0.01). 4. Compared to the negative control group, astragalus polysaccharides and cisplatin could significantly promote human lung cancer A549 cells Bax, Caspase-3 protein expression, whereas inhibition expression of Bcl-2 protein. Compared to the negative control group and single drug group, astragalus the combined effects of polysaccharides with cisplatin effect was stronger, the differences were statistically significant(P<0.01).Conclusions Astragalus polysaccharides and cisplatin inhibit the proliferation of human lung adenocarcinoma A549 cells and promote apoptosis, the effect of which combination significantly enhanced. Astragalus polysaccharides and cisplatin synergistically enhance the pro-apoptotic effects on lung adenocarcinoma A549 cells, and its mechanism may be associated with reduced expression of Bcl-2, up-regulated expression of Bax and Caspase-3.更多还原