【作者】 邱艳君；

【导师】 朱建思；

【作者基本信息】 南华大学 ， 病理学与病理生理学， 2011， 硕士

【摘要】 目的研究白杨素(chrysin, ChR)是否通过上调死亡受体5(Death receptor 5, DR5)增敏肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis-inducing ligand , TRAIL)诱导人肝癌Bel-7402细胞凋亡作用。方法体外培养人肝癌Bel-7402细胞。采用MTT法测定细胞活性。碘化丙啶(PI)染色流式细胞术(FCM)定量分析细胞凋亡率。DNA琼脂糖凝胶电泳确证诱导细胞凋亡作用。Western Blotting检测细胞DR5蛋白的表达。结果MTT比色法测定结果表明,单用ChR或TRAIL处理48小时对人肝癌Bel-7402细胞增殖活性抑制作用的半数抑制浓度(IC50值)分别为252μmol/L和664 ng/mL,然而,用40μmol/L ChR预孵育30分钟后,再加入TRAIL处理48小时, IC50值为91 ng/mL,合并用药效应的CI值是0.497。PI染色FCM分析表明,单用40μmol/L ChR或100 ng/mL TRAIL作用48小时,细胞凋亡率(亚二倍体DNA含量细胞百分率)分别为4.92%±0.38%和6.32%±0.39%,但是,用40μmol/L ChR预孵育30分钟后,再加入100 ng/mL的TRAIL处理48小时,细胞凋亡率为31.3%±2.50%;DR5/Fc(1μg/mL)能够使亚毒性浓度的ChR与TRAIL合用的人肝癌Bel-7402细胞凋亡率从32.4%±0.47%下降到15.2%±0.38%(P<0.05)。DNA琼脂糖凝胶电泳显示,40μmol/L ChR预孵育30分钟后,再加入100 ng/mL的TRAIL处理48小时,在人肝癌Bel-7402细胞,展示出典型DNA梯形条带图谱。Western Blot分析结果发现,ChR以浓度和时间依赖的方式上调Bel-7402细胞DR5表达;DR5嵌合蛋白能够有效对ChR上调Bel-7402细胞DR5表达。结论1.亚毒性浓度的ChR具有增强TRAIL诱导人肝癌Bel-7402细胞凋亡作用。2. ChR敏化TRAIL诱导人肝癌Bel-7402细胞凋亡作用的机制与其上调DR5表达有关。更多还原

【Abstract】 ObjectiveTo investigate whether chrysin(ChR) enhance apoptosis induced by TNF-related apoptosis-inducing ligand(TRAIL) via upregulating the expression of death receptor 5(DR5) in human hepatocellular carcinoma (HCC) Bel-7402 cell line.MethodsHuman hepatocellular carcinoma Bel-7402 cell line was cultured in vitro. The cell viability was evaluated by MTT assay. The percentage of sub-G1 cells was determined by flow cytometry(FCM) using PI fluorescence staining. The characteristic features of cell apoptosis was examined using DNA agarose gel electrophoresis. The expressions of DR5 were analyzed by Western blot.ResultsThe MTT assay showed that IC50 for cell viability inhibition in human hepatocellular carcinoma Bel-7402 cells by ChR and TRAIL alone at 48h were 252μmol/L and 664ng/mL, respectively. However, IC50 by TRAIL treatment for 48h pretreated with ChR(40μmol/L) for 30 minute was 91ng/mL, and the CI value for ChR and TRAIL was 0.497. FCM analysis with PI stainning indicated that the percentage of sub-G1 cells in human hepatocellular carcinoma Bel-7402 cell line by 40μmol/L ChR and 100ng/mL TRAIL alone for 48h were 4.92%±0.38% and 6.32%±0.39%, respectively. However, sub-G1 cell percentage after 100ng/mL TRAIL treatment for 48h pretreated with 40μmol/L ChR for 30 minute was 31.3%±2.50%. Furthermore, human recombinant DR5/Fc chimera protein can result in descend the cell death rate from 32.4%±0.47% to 15.2%±0.38%(P<0.05) in human hepatocellular carcinoma Bel-7402 cell line by 100ng/mL TRAIL treatment for 48h pretreated with 40μmol/L ChR for 30 minute. The ladder band could be shown in DNA agarose gel electrophoresis after treatment with 100ng/mL TRAIL for 48h pretreated with 40μmol/L ChR for 30 minute. Western blot analysis indicated that ChR upregulated expression of DR5 in Bel-7402 cells, in a time- and concentration- dependent manner. Human recombinant DR5/Fc chimera protein could effectively attenuate the up-regulation expression of DR5 by ChR.Conclusion1. Chrysin at subtoxic concentration possess enhancement of TRAIL induced apoptosis of human hepatocellular carcinoma Bel-7402 cells.2. The sensitization of TRAIL induced apoptosis of human hepatocellular carcinoma Bel-7402 cells by ChR is associated with up-regulating the expression of DR5 protien.更多还原