【作者】 张世蘋；

【导师】 蔡云清；

【作者基本信息】 南京医科大学 ， 营养与食品卫生， 2008， 硕士

【摘要】 肝癌是临床上常见的消化系统恶性肿瘤之一,在世界范围内,其发病率呈现上升的趋势。肝癌的发生不仅与细胞的异常增殖有关,亦与细胞凋亡的失衡存在密切关系。大量研究发现黄酮类化合物具有抗肿瘤的作用,其机制主要是通过抑制细胞增殖,诱导细胞凋亡和干预细胞信号转导途径中的蛋白激酶等。甘草素是一种从甘草中提取的一种二氢黄酮单体化合物。国内外研究报道甘草素具有多种生理活性作用,但有关甘草素抗肿瘤作用的研究报道甚少。因此,本课题拟观察甘草素对肿瘤细胞增殖的影响及诱导肿瘤细胞凋亡作用,并通过检测凋亡相关蛋白与酶活性的变化深入探讨甘草素诱导人肝癌SMMC-7721细胞凋亡的分子机理,为进一步研究甘草素的抗肿瘤作用机制提供科学依据。第一部分甘草素抑制肿瘤细胞生长及诱导人肝癌SMMC-7721细胞凋亡的研究目的:探讨甘草素(liquiritigenin)对两株肿瘤细胞生长的抑制作用及对人肝癌SMMC-7721细胞凋亡的诱导作用的影响。方法:本研究采用体外培养人结肠癌Lovo细胞和人肝癌SMMC-7721细胞的方法,通过噻唑蓝(MTT)比色法观察甘草素对肿瘤细胞增殖的影响;Hoechst染色法和电镜观察甘草素诱导SMMC-7721细胞凋亡的形态变化;流式细胞仪检测甘草素诱导SMMC-7721细胞凋亡率的变化情况。结果:甘草素对Lovo结肠癌细胞作用24、48和72h时各剂量组出现生长抑制作用,其最高抑制率分别为15.4%、57.1%和84.7%;对SMMC-7721肝癌细胞作用24、48和72h时,各剂量组都已出现生长抑制作用,最高抑制率分别达到85.6%、94.2%和91.3%,并呈现明显的剂量-效应关系。甘草素0.4mM作用SMMC-7721细胞12、24、48和72h,Hoechst染色观察可见细胞核呈致密浓染,或呈碎块状致密浓染;透射电镜观察可见0.4mM甘草素作用细胞72h后,细胞染色质浓缩、空泡。流式细胞仪检测结果发现,甘草素可诱导SMMC-7721细胞凋亡,并呈时间-剂量效应关系,甘草素剂量为0.6mM作用24h后,即可引起23.52%的细胞发生凋亡。结论:甘草素对人结肠癌Lovo细胞和人肝癌SMMC-7721细胞的生长均有抑制作用,其中对SMMC-7721肝癌细胞的生长抑制作用更加明显,并能诱导SMMC-7721肝癌细胞细胞凋亡。第二部分研究甘草素诱导人肝癌SMMC-7721细胞凋亡的氧化机制目的:探讨甘草素诱导人SMMC-7721肝癌细胞凋亡作用的氧化机制。方法:细胞经氧化敏感的荧光染料2,7-二氢二氯荧光素(DCFH)和罗丹明123(RH-1231)染色后,用流式细胞仪分别测定细胞内氧化水平(ROS)线粒体跨膜电位的变化;黄嘌呤氧化酶法测定SOD活性,5’5’-二硫代二硝基苯甲酸(DTNB)法测定GSH和GSH-PX活性;通过免疫印迹法测定凋亡相关蛋白(p53、Bcl-2和survivin)的表达水平,采用酶标法检测caspase-3酶的活性。结果:0.4mM甘草素作用SMMC-7721细胞后出现ROS积聚,作用3h时,ROS已出现显著升高,并且呈现时间效应关系,作用9h达到最高峰;预先加入NAC作用30min后再加入甘草素,发现NAC可抑制细胞凋亡和细胞内ROS的产生;同时,随着作用时间的延长,线粒体膜电位不断下降,在作用9h和12h时出现显著下降;细胞内抗氧化酶SOD、GSH和GSH-PX活性均在甘草素作用后出现持续下降;0.4mM甘草素作用0、12、24、48和72h后,凋亡相关蛋白p53和Bcl-2表达水平分别呈时间依赖性上调和下调,survivin蛋白和procaspase-3表达下调,caspase-3活性上升。结论:甘草素诱导SMMC-7721肝癌细胞凋亡的机制可能与其促氧化机制有关。更多还原

【Abstract】 Liver cancer is a kind of alimentary system cancers, the morbility of which is increasing in the world. The liver cancer development is not only related with the abnormal generation in cells but also with the disequilibrium in cell apoptosis. Emerging studies have suggested that flavonoids function their potent property of anti-tumor through cell growth inhibitory, induction of cell apoptosis and intervention effects on protien kinase in signal transduction pathway. Liquiritigenin is a flavanone extracted from licorice, possessing a wind range of physiology properties, however, few studies have been reported that liquiritigenin induces apoptosis in tumor cells. In the present study, we plan to investigate the growth inhibitory and pro-apoptotic effects on tumor cells of liquiritigenin; Molecular mechanism of liquiritigenin induce-apoptosis in SMMC-7721 cells was explored by detecting apoptosis-associated proteins and kinases, which was studied to clarify the mechanism of anti-tumor effects induced by liquiritigenin. Objective: The purpose of this study was to investigate the growth inhibitory effect of liquiritigenin on two kinds of tumor cells and explore apoptotic effects of SMMC-7721 cells induced by liquiritigenin. Methods: MTT assay was used to observe the effects on growth inhibition induced by liquiritigenin in Human colorectal cancer Lovo cells and Human hepatocellular carcinoma SMMC-7721 cells. The morphological changes effect of liquiritigenin -induced apoptosis was observed with Hoechst staining and transmission electron microscope, the percentage of apoptotic cells was analyzed by Annexin V-FITC/PI. Results: Liquiritigenin had the inhibitory effects on proliferation of Lovo cells after been treated with liquiritigenin for 24、48 and 72 hours at each concentration, and the high inhibition ratio were 15.4%、57.1% and 84.7%; The high inhibition ratio of liquiritigenin against SMMC-7721 cells was 85.6%, 94.2%,91.3% on 24、48 and 72 hours respectively, and it demonstrated obvious growth inhibitory effect in a dose dependent-manner. Using Hoechst 33258 staining and transmission electron microscope, morphological changes of SMMC-7721 cells induced by 0.4mM liquiritigenin for 72h were found, which exhibited characteristic features of apoptosis including chromatin condensation, nuclear fragmentation and vacuole. Liquiritigenin could induce apoptosis on SMMC-7721 cells in a time-and-dose dependent manner. At 24h, the apoptotic cells enhanced from 7.06% (control) to 23.52% at 0.4mM liquiritigenin. Conclusion: Liquiritigenin has unvarying anti-tumor effects on Human colorectal cancer Lovo cells and Human hepatocellular carcinoma SMMC-7721 cells. It could induce stronger inhibitory effect and apoptotic effect on SMMC-7721 cells.Section 2 Study on apoptosis mechanism of Human hepatocellular carcinoma SMMC-7721 cells induced by liquiritininObjective: The aim of our study was to explore the mechanism of liquiritigenin-induced apoptosis in human hepatocellular carcinoma SMMC-7721 cells. Methods: We used an oxidant-sensitive fluorescent probe, DCFH-DA to examine the production of reactive oxygen species, the mitochondrial membrane potential was investigated with Rh123 and the activity of glutathione (GSH), glutathione peroxidase (GSH-PX), and superoxide dismutase (SOD) were detected by biochemical methods; The levels of p-53, bcl-2, survivin and caspase-3 proteins were detected by western blot. Results: The induction of apoptosis by 0.4mM liquiritigenin was accompanied with the production of reactive oxygen species (ROS), disruption of mitochondrial membrane potential and depletion of antioxidant enzymes. The significant ROS generation was firstly found at 3h and being time-dependent until 9h, while a time-dependent decrease in membrane potential occurred after liquiritigenin treatment and the significant loss appeared at 9h and 12h. Cells were pretreated with N-acetyl-cysteine (NAC), a free radical scavenger, which results provide the evidence that liquiritigenin inducing apoptosis and ROS production were suppressed. Further studies found that the level of p53 protein increased and the Bcl-2 protein decreased in time-dependent manner, the expression levels of survivin and pro-caspase-3 were down-regulated. In addition, a concomitant time-dependent increase in caspase-3 activity was observed by liquiritigenin treatment. Conclusion: These findings suggested that the liquiritigenin inducing apoptosis associated with liquiritigenin as a pro-oxidant in SMMC-7721 cells.更多还原