【作者】 刘日嘉；

【导师】 季宇彬；

【作者基本信息】 哈尔滨商业大学 ， 药理学， 2010， 硕士

【摘要】 本文探讨了藻红蛋白(Phycoerythrin,PE)对体外培养的人乳腺癌MCF-7细胞抗肿瘤作用的研究,并阐述了其调控p53-p21-pRB途径诱导人乳腺癌MCF-7细胞衰老的分子机制。在体外研究中,利用四甲基偶氮唑盐(MTT)比色法,发现藻红蛋白可抑制人乳腺癌MCF-7细胞的生长,抑制效果明显,其IC50为134.87μg·mL-1。通过形态学鉴定藻红蛋白对肿瘤细胞衰老作用,用β-半乳糖苷酶染色试剂盒,DAPI染色液对肿瘤细胞染色,利用普通光学显微镜和荧光显微镜进行观察,发现肿瘤细胞出现典型的衰老特征：肿瘤细胞变大变扁平,细胞皱缩、胞质内空泡形成,并出现发芽起泡现象。经β-半乳糖苷酶(SA-β-gal)染色法染色,倒置显微镜观察,发现给药组细胞分泌衰老特征性β-半乳糖苷酶含量增加,经染料染色,生成蓝色物质。DAPI染色法荧光显微镜观察,给药组细胞核中DNA显示点状聚集分布,并且随着藻红蛋白浓度的递增,现象越来越明显,表明细胞衰老的比例不断增加。随后,利用Fluo-3/AM进行染色,采用激光共聚焦显微镜检测藻红蛋白对肿瘤细胞中钙离子浓度的影响。研究发现,随着藻红蛋白浓度的递增,藻红蛋白能够明显升高肿瘤细胞中钙离子的浓度。且呈现一定的剂量依赖关系。利用TMRE进行染色,采用激光共聚焦显微镜检测藻红蛋白对肿瘤细胞中线粒体膜电位的影响。研究发现,随着藻红蛋白浓度的不断增大,藻红蛋白能够明显降低肿瘤细胞中线粒体膜电位。利用2’,7’-二氯氢化荧光素二脂(DCFH-DA)对肿瘤细胞染色,采用激光共聚焦显微镜检测藻红蛋白对肿瘤细胞中活性氧含量的影响。研究发现,随着藻红蛋白浓度的升高,肿瘤细胞中活性氧的含量也不断增加。在此基础上,我们初步考察了藻红蛋白诱导肿瘤细胞衰老的途径。在激光共聚焦显微镜下观察,不同浓度的藻红蛋白作用人乳腺癌MCF-7细胞48h后,肿瘤细胞内的p53、p21和pRB蛋白的荧光强度均增强,并且随着藻红蛋白浓度的增加,p53、p21和pRB蛋白的荧光强度也随之增强,说明藻红蛋白能诱导人乳腺癌MCF-7细胞表达衰老相关蛋白p53、p21及pRB。综上所述,本课题通过体外实验证明了藻红蛋白对人乳腺癌MCF-7细胞具有明显的抗肿瘤活性。通过形态学观察,藻红蛋白能诱导肿瘤细胞出现典型的衰老特征。在机制作用方面,可能与其可通过提高肿瘤细胞内钙离子浓度和增加肿瘤细胞内活性氧的产生,从而激活细胞衰老的启动蛋白-p53蛋白,从而诱导细胞衰老的发生。总之,藻红蛋白在体外具有明显的抗肿瘤作用且藻红蛋白能通过调控p53-p21-pRB途径诱导人乳腺癌MCF-7细胞衰老。更多还原

【Abstract】 This study explores the actions of the inhibition of human breast cancer cells MCF-7 cultured in vitro by Phycoerythrin (PE) and described its regulation of p53-p21-pRB pathways to induce human breast cancer cells MCF-7 of the molecular mechanism of senescence.In vitro studies using mononuclear cell direct cytotoxicity assay(the MTT method) and found PE inhibit human breast cancer cells MCF-7 growth inhibition effect obviously,with IC50 being 134.87μg·mL-1.By morphological identification of PE senescence effect on the tumor cells, usingβ-galactosidase staining kit, DAPI staining of tumor cells stained, using ordinary optical microscope and fluorescence microscope observations, tumor cells have the typical senescence characteristics, found that great changes of tumor cells become flattened, cell shrinkage, cytoplasmic vacuolization and begin to sprout, blistering. The P-galactosidase (SA-P-gal) staining, inverted microscope and found that treatment group characteristic of senescence cells to secrete P-galactosidase was increased by dyeing to produce blue material. DAPI staining observed by fluorescence microscopy, the nucleus of drug-treated DNA showed punctate aggregated distribution, and with PE concentration increased, the phenomenon of more and more obviously that increasing the proportion of cells senescence.Subsequently, the use of Fluo-3/AM dyed, using confocal laser scanning microscope detection of PE on the tumor cells, calcium ion concentration. The study found that with increasing concentration of PE, PE tumor cells can significantly increase the concentration of calcium ions. And showed a definite dose-dependent relationship. Carried out using TMRE staining, using confocal laser scanning microscope phycoerythrin on the tumor cells, mitochondrial membrane potential impact. The study found that with the PE concentration increasing, PE can significantly reduce the mitochondrial membrane potential in tumor cells. The use of 2’,7’-dichloro fluorescein hydrogenated fat (DCFH-DA) staining of tumor cells, using confocal laser scanning microscope of tumor cells, PE content of reactive oxygen species. The study found, with the PE concentration increased, the tumor cells of reactive oxygen species levels are also increasing. On this basis, we initially examined the PE inducing tumor cells senescence way. In the confocal laser scanning microscope observation, different concentrations of PE effect on human breast cancer cells MCF-7 48h after the tumor cells intracellular p53, p21, and pRB proteins have increased fluorescence intensity and concentration with the PE increase, p53, p21, and pRB protein in the fluorescence intensity also increasesd. This effect shows PE can induce human breast cancer cells MCF-7 expressed senescence-associated protein, p53, p21, and pRB. From the above, it can be concluded that PE can inhibit in vitro the growth of human breast cancer cells MCF-7. By morphological observation, tumor cells can be induced by PE of the typical senescence characteristics. Role in the mechanism may be related to tumor cells by increased intracellular calcium concentration and raised the ROS level, which activate cells senescence start-protein-p53 protein, thereby inducing cells senescence occurred. In a word, PE has the anti-tumor effect obviously in vitro and PE can induce the human breast cancer cells MCF-7 senescence via p53-p21-pRB pathways.更多还原