盐霉素联合17-AAG对胃癌细胞体外增殖的影响

发布时间：2018-01-20 04:11

本文关键词： 盐霉素胃癌 Caspase-3 NF-κB 凋亡　出处：《延安大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:探讨盐霉素单独及联合17-AAG对人胃癌细胞株SGC-7901体外增殖的影响,旨在明确盐霉素单独及联合17-AAG对人胃癌细胞体外增殖抑制作用及其机制。方法:以人胃癌SGC-7901细胞株为研究对象,应用MTT比色法检测盐霉素单独及联合17-AAG对胃癌细胞的增殖抑制率;倒置相差显微镜观察细胞形态变化;荧光显微镜观察经PI及AO染色后细胞凋亡形态变化;流式细胞术检测细胞周期及凋亡率的变化;Western blotting检测Caspase-3蛋白表达的变化;免疫细胞化学法检测NF-κBp65、FAS-L蛋白表达的变化;采用SPSS22.0软件进行统计分析,所有分析数据均用x±s表示,所有均数间的比较用单因素方差分析,多个均数间的多重比较用LSD-t检验,以P0.05为差异具有统计学意义。结果:1.MTT结果显示盐霉素在一定的浓度范围(1、2、4、8、16、32μmol/L)内分别作用24h、48h、72 h可以有效抑制胃癌SGC-7901细胞的增殖(P0.05或P0.01)。抑制效果呈剂量和(或)时间的依赖性。盐霉素(4、8、16、32μmol/L)联合17-AAG(0.625μmol/L)可以有效抑制胃癌细胞的增殖(P0.05),且两药联合抑制效果显著高于单独用药组(P0.05),提示盐霉素可以增加17-AAG对胃癌细胞的敏感性。且联合作用效果也呈浓度和时间依赖性。2.倒置相差显微镜及荧光显微镜观察可见典型的细胞凋亡形态改变。3.流式细胞仪检测细胞周期,结果显示4、8、16μmol/L盐霉素使胃癌细胞周期阻滞在S期,它们所占细胞周期的比例分别为26.90%、34.57%、41.83%,与对照组比较差异有统计学意义(P0.05)。8μmol/L盐霉素与0.625μmol/L17-AAG单独及联合均导致细胞阻滞在S期,S期比例分别为34.57%、26.92%、40.10%显著高于对照组的23.52%(P0.05),联合用药组与单独用药组比较差异也具有统计学意义(P0.05)。4.流式细胞仪检测细胞凋亡,结果显示4、8、16μmol/L盐霉素使胃癌细胞周期阻滞在S期,它们所占细胞周期的比例分别为26.90%、34.57%、41.83%,与对照组比较差异有统计学意义(P0.05)。8μmol/L盐霉素与0.625μmol/L17-AAG单独及联合均导致细胞阻滞在S期,S期比例分别为34.57%、26.92%、40.10%显著高于对照组的23.52%(P0.05),联合用药组与单独用药组比较差异也具有统计学意义(P0.05)。5.结果显示:4、8、16μmol/L盐霉素作用胃癌SGC-7901细胞48h后,细胞的凋亡率分别为10.52%、14.69%、20.46%,与对照组的3.42%比较,差异有统计学意义(P0.05)。8μmol/L盐霉素与0.625μmol/L17-AAG单独及联合用药后细胞的凋亡率分别为14.69%、7.96%、26.87%,与对照组比较差异均有统计学意义(P0.05),联合用药组与单独用药组比较差异也具有统计学意义(P0.05)。6.Western bloting检测显示盐霉素和17-AAG单独用药均能显著上调SGC-7901细胞中的Caspase-3的表达,且两药联合应用时比单独用药更为明显。7.免疫细胞化学法检测显示盐霉素单独及与17-AAG联合均能显著下调NF-κBp65的表达,显著上调Fas-L的表达,且联合作用蛋白表达变化较单独用药时明显。结论:1.盐霉素能有效抑制人胃癌细胞SGC-7901的体外增殖,其作用呈剂量和时间依赖性;2.盐霉素与17-AAG联合应用可以抑制人胃癌细胞SGC-7901的体外增殖,其作用呈剂量和时间依赖性;3.盐霉素单独及与17-AAG联合均可以诱导胃癌细胞SGC-7901的凋亡,其作用机制可能与上调胃癌细胞内Caspase-3、FAS-L和下调NF-κBp65的表达有关。
[Abstract]:Objective: To investigate the effects of salinomycin alone and combined with 17-AAG on the proliferation of human gastric cancer cell line SGC-7901 in vitro, in order to clear the inhibitory effect and mechanism of salinomycin alone and combined with 17-AAG on the proliferation of human gastric cancer cells in vitro. Methods: human gastric cancer cell line SGC-7901 as the research object, should be detected by MTT inhibition rate by salt colorimetric method in separate and combined with 17-AAG on proliferation of gastric cancer cells; morphologic changes were observed under inverted microscope; fluorescence microscopy to observe the changes of apoptosis and morphology of PI and AO staining; detection of cell cycle and apoptosis rate by flow cytometry; changes of Western blotting expression of Caspase-3 protein was detected; detection of NF- kappa Bp65 immunocytochemical method and the alteration of FAS-L expression; SPSS22.0 software was used for statistical analysis, analysis of all data were expressed by X + s, compared with the number of all the single factor variance analysis, multiple mean The multiple comparison by LSD-t test, with P0.05 as the difference was statistically significant. Results: 1.MTT results showed that salinomycin in a certain range of concentrations (1,2,4,8,16,32 mol/L) in 24h 48h 72, respectively, h can effectively inhibit the proliferation of SGC-7901 (P0.05 or P0.01). The inhibitory effect was dose and (or time dependent). Salinomycin (4,8,16,32 mol/L) and 17-AAG (0.625 mol/L) can effectively inhibit the proliferation of gastric cancer cells (P0.05), and the two drugs combined with inhibitory effect was significantly higher than the single drug group (P0.05), suggesting that salinomycin can increase the sensitivity of 17-AAG on gastric cancer cells and the combined effect. It was time and concentration dependent.2. inverted microscope and fluorescence microscope observation showed typical apoptosis morphology change of.3. cell cycle by flow cytometry, the results showed that 4,8,16 mol/L salinomycin to gastric cancer cell cycle arrest In the S period, their proportion of cell cycle were 26.90%, 34.57%, 41.83%, there was statistically significant difference compared with control group (P0.05).8 mol/L Salinomycin and 0.625 mol/L17-AAG alone and in combination resulted in cell cycle arrest at S phase S phase ratio were 34.57%, 26.92%, 40.10% was significantly higher than that of control group of 23.52% (P0.05), difference between combined treatment group and single medication group was statistically significant (P0.05) apoptosis detection by flow cytometry showed that.4., 4,8,16 mol/L the salinomycin gastric cancer cell cycle arrest in S phase, the proportion of cell cycle were 26.90%, 34.57%, 41.83%, there are significant differences compared with the control group (P0.05).8 mol/L Salinomycin and 0.625 mol/L17-AAG alone and in combination resulted in cell cycle arrest at S phase S phase ratio were 34.57%, 26.92%, 40.10% higher than that of the control group 23.52% (P0.05), combination group Compared with the single drug group was also statistically significant difference (P0.05).5. showed that 4,8,16 mol/L salinomycin effect SGC-7901 gastric cancer cell 48h, cell apoptosis rates were 10.52%, 14.69%, 20.46%, compared with 3.42% in the control group, the difference was statistically significant (P0.05).8 mol/L of Salinomycin and 0.625 mol/L17-AAG alone and combination after the cell apoptosis rates were 14.69%, 7.96%, 26.87%, and the control group had significant difference (P0.05). The difference between the combined group and single drug group was also statistically significant (P0.05).6.Western bloting showed that Salinomycin and 17-AAG alone could up regulate the expression of SGC-7901 Caspase-3 in the cells, and the combination of the two drugs is more obvious than the sole use of detection.7. immunocytochemistry showed salinomycin alone and in combination with 17-AAG could significantly downregulate NF- kappa Bp65 table Up, up regulate the expression of Fas-L, and the joint effect of protein expression than either drug alone was 1.. Conclusion: the proliferation of salinomycin can inhibit the growth of human gastric cancer cells SGC-7901 in vitro. The effect was dose and time dependent proliferation; 2. salinomycin combined with 17-AAG can inhibit human gastric cancer cells SGC-7901 in vitro. The effect was dose and time dependent; 3. salinomycin alone and in combination with 17-AAG could induce apoptosis of gastric cancer cell SGC-7901 and its mechanism may be related to the upregulation of Caspase-3 in gastric cancer cells, FAS-L and expression of NF- kappa Bp65.

【学位授予单位】：延安大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.2

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