【作者】 李润青；

【导师】 单保恩； 马洪；

【作者基本信息】 河北医科大学 ， 临床检验诊断学， 2007， 硕士

【摘要】 目的:研究槲皮素对人结肠癌细胞SW480增殖、凋亡、细胞周期的影响,建立Wnt/β-catenin信号转导通路报告基因模型,研究槲皮素对SW480细胞Wnt/β-catenin信号转导的调控作用和对Wnt/β-catenin信号转导通路相关基因的影响。探讨槲皮素抗肿瘤作用机理,为槲皮素作为抗肿瘤药物开发提供理论依据。方法:1.采用MTT比色法检测槲皮素对人结肠癌细胞SW480细胞增殖反应的作用;2.用流式细胞技术分析槲皮素对人结肠癌细胞SW480细胞周期、凋亡的影响。3.采用Western blot分析槲皮素对SW480细胞cyclin B1蛋白表达的影响。4.建立Wnt/β-catenin信号转导通路报告基因模型,研究槲皮素对SW480细胞Wnt/β-catenin信号转导通路的调控作用。5.用半定量RT-PCR和Western blot方法研究槲皮素对Wnt/β-catenin信号转导通路β-catenin/TCF下游靶基因cyclin D1和survivin mRNA及蛋白表达的影响。6.统计学方法:所有数据采用SPSS 12.0软件进行统计学处理。实验结果均以x±S表示,组内比较采用单因素方差分析,多个实验组均数与一个对照组均数比较用Dunnett-t检验,回归方程间比较用t检验与方差分析,以α=0.05或α=0.01为检验水准。结果:1.槲皮素抑制SW480细胞增殖MTT检测结果显示,40、80、160μmol/L的槲皮素对SW480细胞增殖有明显的抑制作用,与对照组比较有显著性差异(P<0.01),高浓度槲皮素组与低浓度组比较,对SW480细胞抑制作用也明显差异(P<0.05),抑制作用呈剂量依赖性和时间依赖性(r=0.99, P=0.00)。2.槲皮素可诱导SW480细胞周期阻滞和细胞凋亡流式细胞术检测结果显示,40、60、80μmol/L的槲皮素作用SW480细胞48 h后,G0/G1期和S期细胞减少,G2/M期细胞显著增多(P<0.01)。槲皮素可以剂量依赖性地诱导SW480细胞凋亡,80μmol/L的槲皮素作用48 h后,细胞凋亡率(13.32%±4.62%)明显升高,与对照组(2.68%±1.04%)相比有显著性差异(P<0.01)。3.槲皮素可明显下调SW480细胞cyclin B1蛋白的表达经槲皮素作用48 h后,SW480细胞cyclin B1蛋白表达水平明显下降(P<0.05),抑制作用呈剂量依赖性(r=0.999, P=0.001)。4.槲皮素可以明显下调SW480细胞TCF-4报告基因TOPFLASH活性应用双荧光素酶报告基因质粒TOPFLASH/FOPFLASH及内参照质粒pRL-null瞬时转染SW480细胞,建立了Wnt/β-catenin信号转导通路双荧光素酶报告基因模型。经分析结果显示,槲皮素处理转染报告基因的SW480细胞24 h后,TCF-4报告基因TOPFLASH活性显著降低,160μ?mol/L槲皮素作用24 h后,TOPFLASH报告基因RLU值由对照组的195.65±12.25降至11.54±5.60,差异有统计学意义(P<0.05),但FOPFLASH活性没有明显的变化(P>0.05),提示槲皮素可剂量依赖性抑制β-catenin/TCF的转录活性。5.槲皮素可显著下调SW480细胞cyclin D1和survivin mRNA表达和蛋白表达水平60、120μmol/L槲皮素作用24 h后,SW480细胞cyclin D1和survivin mRNA表达和蛋白表达水平均显著下降,抑制作用呈剂量依赖性(P<0.05)。结论:槲皮素可明显抑制SW480细胞增殖,诱导细胞凋亡,将SW480细胞周期阻滞于G2/M期。槲皮素可剂量依赖性抑制β-catenin/TCF的转录活性,下调β-catenin/TCF下游靶基因cyclin D1和survivin的mRNA表达和蛋白表达水平,提示其抗肿瘤机制可能与其抑制Wnt/β-catenin信号转导通路有关。更多还原

【Abstract】 Objective: Effects of quercetin on proliferation, cell cycle and apoptosis of colon carcinoma cell line SW480 were investigated. To investigate the regulation effect of quercetin on the Wnt/β-catenin signaling transcription, a Wnt/β-catenin signaling pathway reporter gene model was build, and the influence of quercetin on mutuality gene of Wnt/β-catenin signaling pathway was studied. By these analysis, the probable molecular mechanisms of quercetin on SW480 cell was analyzed, and maybe provide some theoretical evidences for developing quercetin as an anti-tumor drug.Methods:1. Effect of quercetin on proliferation of SW480 cells was analyzed by MTT assay.2. Cell cycle and apoptosis rate of SW480 cells induced with quercetin were detected by flowcytometry.3. After treatment with quercetin, protein expression of cyclin B1 in SW480 cells was analyzed by Western blot analysis.4. We Build a Wnt/β-catenin signaling pathway reporter gene model. The regulation effect of quercetin on the Wnt/β-catenin signaling transcription was investigated by using the reporter gene model.5. Effects of quercetin on expression ofβ-catenin/TCF downstream target genes, cyclin D1 and survivin, of Wnt/β-catenin signaling pathway were detected by Semi-quantitative RT-PCR and Western blot analysis.6. Statistics Methods: Statistic analysis was performed with SPSS 12.0. All experiment data were indicated Mean±STD.Results:1. Quercetein can inhibite the proliferation of SW480 cells By MTT analysis, it is shown that proliferation of SW480 cells was significantly inhibited by quercetein at concentrations of 40, 80, 160μmol/L (P < 0.01), and showing a dose- and time-dependent manner.2. After treatment with 40, 60, 80μmol/L quercetin for 48 h, the percentages of SW480 cells at G0/G1 phase were decreased, and those at G2/M phase were increased markedly (P<0.01). After incubated with 80μmol/L quercetin for 48 h, apoptosis rate of SW480 cells increased from 2.68%±1.04% of control group to 13.32%±4.62% (P<0.01).3. Expression of cyclin B1 in SW480 cells could be down-regulated by quercetin in a dose-depedent manner (r=0.999, P=0.001).4. Quercetin can down-regulate TCF-4 reporter gene We built a Wnt/β-catenin signaling pathway reporter gene model. Activities of TCF-4 reporter gene was significantly down-regulated by quercetin analyzed by using the reporter gene model (P<0.05), and showing a dose- and time- dependent manner.5. Expression of cyclin D1 and survivin in SW480 cells were down-regulated markedly by quercetin in a dose-dependent manner.Conclusions: Quercetin can inhibit proliferation, induce apoptosis and block the cells at G2/M phase of SW480 cells. Cyclin D1 and survivin expression,β-catenin/TCF downstream target genes, in both mRNA and protein level could be down-regulated markedly by quercetin. The anti-tumor mechanism of quercetin may be related to the inhibition of Wnt/β-catenin signaling pathway.更多还原