【作者】 韩守恒；

【导师】 庄英帜； 朱建思；

【作者基本信息】 南华大学 ， 肿瘤学， 2009， 硕士

【摘要】 目的研究白杨素是否通过抑制NF-kapppaB活性增强TRAIL对人胃癌SGC-7901细胞毒性作用。方法体外培养人胃癌SGC-7901细胞和人胃黏膜GES-1细胞。MTT比色法测定细胞活性。碘化丙啶(PI)染色流式细胞术(FCM)定量分析细胞凋亡程度。DNA琼脂糖凝胶电泳确证诱导细胞凋亡作用。Western Blot检测细胞NF-kappaB(p65)蛋白和磷酸化IκBα蛋白表达。结果MTT比色测定结果显示：单独应用白杨素或TRAIL作用48小时对人胃癌SGC-7901细胞增殖活性抑制作用的半数抑制浓度(IC50值)分别为134μmol/L和402ng/mL,然而,用40μmol/L的白杨素预孵育30分钟后,再加入TRAIL处理48小时,IC50值为47ng/mL,合并用药效应的CI值是0.4676；单独应用白杨素和TRAIL作用48小时或者用40μmol/L的白杨素预孵育30分钟后,再加入TRAIL处理48小时,对永生化二倍体人胃粘膜GES-1细胞增殖活性抑制的IC50值分别为747μmol/L、1348ng/mL和845ng/mL,合并用药效应的CI值是1.7579。碘化丙啶(PI)染色流式细胞术(FCM)分析结果表明：单独应用白杨素40μmol/L或TRAIL100ng/mL作48小时,亚二倍体DNA含量细胞百分率分别为4.65%±0.58%和3.60%±0.16%；但是,用40μmol/L的白杨素预孵育30分钟后,再加入100 ng／mL的TRAIL处理48小时,亚二倍体DNA含量细胞百分率为49.87%±4.27%；单独应用白杨素40μmol/L和TRAIL100ng/mL作用48小时,或者用40μmol/L的白杨素预孵育30分钟后,再加入100 ng/mL的TRAIL处理48小时,永生化二倍体人胃粘膜GES-1细胞的亚二倍体DNA含量细胞百分率分别为3.09%±0.24%、4.11%±0.22%和4.34%±0.095%,与溶媒组(2.19%±0.06%)比较差异无统计学显著性意义。DNA琼脂糖凝胶电泳显示：用40μmol/L的白杨素预孵育30分钟后,再加入100ng/mL的TRAIL处理人胃癌SGC-7901细胞48小时,展示出典型DNA梯形条带图谱。Western Blot分析结果发现：白杨素以浓度和时间依赖的方式抑制SGC-7901细胞NF-κB(p65)蛋白表达；并以浓度和时间依赖的方式下降SGC-7901细胞IκBα蛋白磷酸化水平。结论1.亚细胞毒性浓度的白杨素具有增强TRAIL对人胃癌SGC-7901细胞毒性作用。2.亚细胞毒性浓度的白杨素增强TRAIL对人胃癌SGC-7901细胞毒性作用的机制与降低细胞IκBα蛋白磷酸化水平和抑制NF-κB(p65)蛋白表达有关。更多还原

【Abstract】 Objective To investigate whether chrysin(ChR) enhance cytotoxicity induced by recombinant human solubility TNF-related apoptosis-inducing ligand(TRAIL) through inhibiting NF-κB activity in human gastric cancer SGC-7901 cell line.Methods Human gastric cancer SGC-7901 cell line and human gastric mucosa GES-1 cell line were cultured in vitro. The cell viability was determined using MTT assay. The sub-G1 cell percentage was examined by flow cytometry using PI fluorescence staining. The characteristic features of cell apoptosis was certified by DNA agarose gel electrophoresis. The expressions of phosphorylated IκBαand NF-κB(p65) protein were analyzed by Western blot.Results The MTT assay showed that IC50 of cell viability inhibition in human gastric cancer SGC-7901 cells by ChR and TRAIL alone for 48h were 134μmol/L and 402ng/mL, respectively. However, IC50 by TRAIL treatment for 48h pretreated with ChR(40μmol/L) for 30 minute was 47ng/mL, and the CI value for ChR and TRAIL was 0.4676. IC50 of cell viability inhibition in immortalization dipoid human gastric mucosa GES-1 cells by ChR and TRAIL alone for 48h or TRAIL treatment for 48h pretreated with ChR(40μmol/L) for 30 minute were 747μmol/L,1348ng/mL and 845ng/mL, respectively. The CI value for ChR and TRAIL was 1.7579. Flow cytometry(FCM) analysis with PI stainning indicated that the sub-G1 cell percentage in human gastric cancer SGC-7901 cells by ChR(40μmol/L) and TRAIL(100ng/mL) alone for 48h were 4.65%±0.58% and 3.60%±0.16%, respectively. However, sub-Gl cell percentage after TRAIL (100ng/mL) treatment for 48h pretreated with ChR(40μmol/L) for 30 minute was 49.87%±4.27%. The sub-G1 cell percentage in human gastric mucosa GES-1 cells by ChR(40μmol/L) and TRAIL(100ng/mL) alone for 48h or TRAIL(100ng/mL) treatment for 48h pretreated with ChR(40μmol/L) for 30 minute were 3.09%±0.24%,4.11%±0.22%, and 4.34%±0.095%, respectively. There was not significant difference in comparison with solvent group(2.19%±0.06%). The ladder band could be shown in DNA agarose gel electrophoresis after treatment with TRAIL(100ng/mL) for 48h pretreated with ChR(40μmol/L) for 30 minute in human gastric cancer SGC-7901 cells. Western blot analysis indicated that ChR inhibited expression of NF-κB(p65) protein and depressed the phosphorylation level of IκBαprotein in SGC-7901 cells, in a time-and concentration-dependent manner.Conclusion1. Chrysin at suboptimal concentration possess augmentation of TRAIL induced cytotoxicity of in human gastric cancer SGC-7901 cells.2. The potentialization of TRAIL induced cytotoxicity of human gastric cancer SGC-7901 cells by ChR is associated with inhibiting the expression of NF-κB(p65) protein and depressing the phosphorylation level of IκBαprotein.更多还原