双氢青蒿素联合紫杉醇对人胃癌MGC-803细胞增殖和凋亡的影响

发布时间：2018-06-21 01:37

本文选题：双氢青蒿素 + 紫杉醇　；参考：《南华大学》2016年硕士论文

【摘要】：目的:以人胃癌MGC-803细胞为研究对象,观察双氢青蒿素与紫杉醇对该细胞增殖和凋亡的影响,以初步探讨两药联合是否能起到协同作用,从而为胃癌的化疗提供新思路。方法:体外培养MGC-803细胞,采用CCK-8法于24h分别检测不同浓度双氢青蒿素、紫杉醇及两药联合对细胞增殖的影响,并用CHOU-Talalay法评估两药联合的效果;用流式细胞仪分析各药物组对细胞周期阻滞的影响;用AO/EB染色剂处理细胞后,在荧光显微镜下观察各药物处理组作用MGC-803细胞后的凋亡情况;用逆转录聚合酶链反应法和免疫蛋白印迹法检测对人胃癌MGC-803细胞的Bcl-2、Bax和Caspase-3表达的影响。结果:1、双氢青蒿素(191.082、955.410、4777.048、23885.240、119426.202ng/ml)、紫杉醇(2.4、12、60、300、1500ng/ml)及两药联合(191.082+2.4、955.410+12、4777.048+60、23885.240+300、119426.202+1500ng/ml)对人胃癌MGC-803细胞均具有增殖抑制作用并呈浓度依赖(p0.05);联合用药组的增殖抑制作用优于单一用药组(p0.05),其中两药小剂量联合用药时呈协同作用,大剂量联合用药时呈拮抗作用。2、通过流式细胞仪对药物处理后细胞的周期检测,我们发现双氢青蒿素(955.410ng/ml)作用后出现G0/G1期、S期细胞阻滞(p0.001),紫杉醇(12ng/ml)作用后出现G2/M期细胞阻滞(p0.001),两药联合(DHA955.410ng/ml+PTX12ng/ml)表现出S期、G2/M期阻滞(p0.001);联合用药组相比于紫杉醇用药组有G2/M期细胞阻滞减少(p0.001)。3、荧光显微镜观察发现,双氢青蒿素(955.410ng/ml)、紫杉醇(12ng/ml)处理MGC-803细胞24h后出现较多橙黄及橙红色细胞,相比于绿色细胞而言,体积缩小、胞质浓缩,具有凋亡细胞的明显特征。同样条件下,联合组的凋亡细胞数较单药组明显增多,且晚期凋亡细胞数目居多。4、双氢青蒿素(955.410ng/ml)及紫杉醇(12ng/ml)作用于人胃癌MGC-803细胞后均能促进Bax、Caspase-3的表达,抑制Bcl-2的表达,而两药联合(DHA955.410ng/ml+PTX12ng/ml)表现出更明显的效果,差异均有统计学意义(p0.05)。结论:1、双氢青蒿素、紫杉醇对人胃癌MGC-803细胞的增殖抑制呈浓度依赖;2、两药联合对MGC-803细胞有增殖抑制作用,它们合用时小剂量是协同作用,而大剂量则为拮抗作用;3、两药均可通过增加Bax、Caspase-3的表达及减少Bcl-2的表达来诱导细胞凋亡。
[Abstract]:Objective: to observe the effects of dihydroartemisinin and paclitaxel on the proliferation and apoptosis of human gastric cancer cell line MGC-803 in order to explore whether the combination of the two drugs can play a synergistic effect and provide a new way for the chemotherapy of gastric cancer. Methods: MGC-803 cells were cultured in vitro. The effects of dihydroartemisinin, paclitaxel and the combination of two drugs on the proliferation of MGC-803 cells were detected by CCK-8 method at 24 hours, and the effects of the two drugs were evaluated by CHOU-Talalay method. The cell cycle arrest was analyzed by flow cytometry, and the apoptosis of MGC-803 cells treated with AO-EB staining was observed under fluorescence microscope. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression of Bcl-2P Bax and Caspase-3 in human gastric cancer MGC-803 cells. Results: 1, 191.082955.41010, 4777.048, 23885.240, 19426.2026 ng / ml, paclitaxel 2.4126060300 / 1500ng / ml) and two drugs combined, 191.082 2.4955.410, 121.082 2.4955.410, 121.082 2.4955.410, 121.082 2.4955.410, 121.082 2.4955.410, 23887.048 60,23887.048 60,19426.202 1500ng / ml) all had inhibitory effects on the proliferation of human gastric cancer MGC-803 cells in a concentration-dependent manner, and the inhibitory effect of the combined treatment group was superior to that of the single drug group (p0.05N), two of which were found to be in a dose-dependent manner. There was a synergistic effect when the drug was combined with small dosage. The cell cycle after drug treatment was detected by flow cytometry. We found that after the treatment of dihydroartemisinin (955.410ng / ml), the cell block of G _ 0 / G _ 1 phase in S phase was found, and that of paclitaxel 12ng / ml group showed G _ 2 / M phase cell block P _ (0.001), and the combination of two drugs, DHA 955.410ng / ml PTX _ (12ng / ml), showed S phase G _ 2M phase block (P _ 0.001), compared with paclitaxel group (P _ 0.001). There was a decrease in G _ 2 / M phase arrest, P 0.001 ~ (-1) ~ 3, and fluorescence microscopy showed that, MGC-803 cells treated with dihydroartemisinin 955.410 ng / ml, paclitaxel 12ng / ml for 24 h showed more orange and orange red cells. Compared with green cells, the cells were smaller in volume and concentrated in cytoplasm and had the obvious characteristics of apoptotic cells. Under the same conditions, the number of apoptotic cells in the combination group was significantly higher than that in the single drug group, and the number of apoptotic cells in the late stage was mainly .4.The expression of BaxCaspase-3 was inhibited and the expression of BaxCaspase-3 was inhibited by the addition of dihydroartemisinin 955.410ng / ml and taxol 12ng / ml to human gastric cancer MGC-803 cells. The combination of DHA 955.410ng / ml PTX 12ng / ml showed a more obvious effect, and the difference was statistically significant (P 0.05). Conclusion the inhibition of proliferation of human gastric cancer MGC-803 cells by 1: 1, dihydroartemisinin and paclitaxel is concentration-dependent. The combination of the two drugs can inhibit the proliferation of MGC-803 cells. However, the antagonistic effect of high dose was antagonistic. Both drugs could induce apoptosis by increasing the expression of Baxton-Caspase-3 and decreasing the expression of Bcl-2.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.2

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