姜黄素诱导胃癌细胞SGC-7901凋亡机制的研究

发布时间：2018-02-25 15:19

本文关键词： 姜黄素 IAPS 信号通路胃癌细胞细胞凋亡　出处：《南华大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:通过观察姜黄素对人胃癌细胞SGC-7901生长的影响、细胞周期改变及NF-κB、Livin、Caspase-3表达的变化,初步探讨姜黄素诱导胃癌细胞SGC-7901凋亡的可能机制。方法:(1)将对数生长期的SGC-7901细胞,按每孔1000细胞加入到96孔细胞培养板中,接种容积为100μl;37℃、5%CO2条件下预贴壁12h后,分别加入浓度为10、20、40、60、80、100μmol/L姜黄素溶液、DMSO溶剂对照组及培养基空白对照组各100μl,每个浓度设置5个重复孔,分别培育12h、24h、36h、48h、72h后,用CCK-8法筛选最佳药物浓度及作用时间;(2)设置空白对照组及20μmol/L,40μmol/L,60μmol/L姜黄素浓度组,将对数生长期的SGC-7901细胞,按每孔5000细胞加入到6孔细胞培养板中,待细胞数量达到106/孔后,分别加入2m L1640培养基、20、40、60μmol/L姜黄素溶液,培育24h,用流式细胞仪测出各组细胞的细胞周期和凋亡率;(3)选取空白对照组及20μmol/L,40μmol/L,60μmol/L姜黄素浓度组,提取各组细胞的蛋白,Western Blot检测各组SGC-7901细胞中NF-κB、Livin及Caspase-3等与胃癌细胞内抗凋亡与促凋亡蛋白表达量的变化。(4)本实验所有实验结果采用SPSS18.0统计并进行分析,对照组与实验组采用单因素配对样本的t检验,方差分析两组及两组以上数据结果。结果:(1)CCK-8检测结果:姜黄素药物浓度增加以及作用时间的延长,SGC-7901细胞的增殖活力值逐渐下降,与对照组比较差异具有显著性(P0.05)。Pearson关联性分析结果显示:与对照组比较SGC-7901细胞对不同浓度姜黄素具有剂量的依赖性(r=0.901,P=0.000.01)以及时间的依赖性(r=0.389,P=0.000.01)。(2)流式细胞仪检测胃癌SGC-7901细胞的周期:姜黄素作用于SGC-7901细胞的药物浓度的增加以及时间的延长,处于G1期的SGC-7901细胞数目明显增加,且S期的细胞数目均下降。FCM检测20μmol/L、40μmol/L、60μmol/L姜黄素溶液对SGC-7901凋亡率较对照组差异具有显著性(P0.05)。(3)Western Blot免疫印迹分析蛋白表达,加入姜黄素药物处理SGC-7901细胞,随着姜黄素浓度的增高,SGC-7901细胞内NF-κB蛋白同Livin蛋白表达水平逐渐降低,Caspase-3蛋白表达水平逐渐升高。结论:1:姜黄素可明显抑制体外培养的人胃癌SGC-7901细胞的增殖活力,促进胃癌细胞凋亡。其抑制细胞增殖与促进细胞凋亡具有明显的浓度及时间依赖关系;2:姜黄素可能影响胃癌SGC-7901细胞周期,并阻滞在细胞周期G1期;3:姜黄素可能通过下调SGC-7901细胞内NF-κB信号转导蛋白及Livin蛋白表达水平,激活Caspase-3蛋白表达,促进SGC-7901细胞凋亡。
[Abstract]:Objective: the effect on the growth of human gastric cancer cell SGC-7901 by observing the changes of cell cycle and curcumin, NF- kappa B, Livin, Caspase-3 expression changes, discuss the possible mechanism of curcumin induced apoptosis of gastric cancer cell line SGC-7901. Methods: (1) the SGC-7901 cells in logarithmic growth phase at 1000 cells per well was added to the 96 hole cell culture plate, inoculation volume of 100 mu L; 37 DEG C, 5%CO2 under the condition of pre adherent after 12h were added at the concentration of 10,20,40,60,80100 mol/L curcumin solution, DMSO solvent control group and medium control group 100 L, each concentration set 5 repeated holes, respectively 24h, 36h, 12h training. 48h, 72h, the best screening time of drug concentration and action by CCK-8 method; (2) blank control group and 20 mol/L, 40 mol/L, 60 mol/L curcumin concentration group, the SGC-7901 cells in logarithmic growth phase at 5000 cells per well into the 6 Hole cell culture plate The number of cells, to achieve 106/ hole, we added the 2m L1640 medium, 20,40,60 mol/L curcumin solution, cultivation of 24h, using flow cytometry to measure cell apoptosis rate and cell cycle; (3) select the blank control group and 20 mol/L, 40 mol/L, 60 mol/L curcumin concentration group the cells were extracted, the protein of each group of SGC-7901 cells in Blot detection of Western NF- kappa B, and Caspase-3 and Livin in gastric cancer cells apoptosis and apoptosis protein expression. (4) in this experiment, all the experimental results using SPSS18.0 and statistical analysis, t test of control group and experimental group with single factor pair analysis of two groups of samples, and more than two sets of data variance. Results: (1) CCK-8 test results: the increase of curcumin concentration and the prolongation of time, the viability of SGC-7901 cells decreased, with significant differences compared with the control group (P0.0 5).Pearson correlation analysis showed that: compared with the control group of SGC-7901 cells in a dose dependent on different concentrations of curcumin (r=0.901, P=0.000.01) and time dependent (r=0.389, P=0.000.01). (2) flow cytometry SGC-7901 gastric cancer cell cycle: the increase of drug concentration in SGC-7901 cells by curcumin the role of the time and in the number of SGC-7901 cells in G1 phase increased significantly, and the number of cells in S phase decreased.FCM detection of 20 mol/L, 40 mol/L, 60 mol/L curcumin solution on the apoptosis rate of SGC-7901 was significant difference compared with the control group (P0.05). (3) the expression of Western Blot protein by Western blot analysis curcumin, drug treatment of SGC-7901 cells, with the increase of the concentration of curcumin in SGC-7901 cells, NF- kappa B protein and Livin protein expression decreased, Caspase-3 protein expression level increased gradually. Conclusion: ginger 1: Huang Suke significantly inhibited the in vitro cultured human gastric cancer SGC-7901 cell proliferation, promote apoptosis of gastric cancer cells. The inhibition of cell proliferation and promote cell apoptosis in a dose and time dependence of 2: obviously; curcumin may affect gastric cancer SGC-7901 cell cycle and arrest in the G1 phase of the cell cycle; 3: curcumin by inhibiting the expression of NF- B signal transduction protein and Livin protein level in SGC-7901 cells, activate the expression of Caspase-3 protein and promote the apoptosis of SGC-7901 cells.

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

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