藻红蛋白调控p53-p21-pRB途径诱导MCF-7细胞衰老的研究

发布时间：2018-08-23 10:12

【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。
[Abstract]:In this paper, the antitumor effect of phycoerythrin (PE) on human breast cancer MCF-7 cells in vitro was studied, and the molecular mechanism of its regulation of p53-p21-pRB pathway on the senescence of human breast cancer MCF-7 cells was discussed. In vitro, phycoerythrin was found to inhibit the growth of human breast cancer MCF-7 cells by tetramethyl azolium salt (MTT) colorimetry. The inhibitory effect of phycoerythrin on the growth of human breast cancer MCF-7 cells was significant, and its IC50 was 134.87 渭 g ml -1. The aging effect of phycoerythrin on tumor cells was identified by morphology. The tumor cells were stained with 尾 -galactosidase staining kit DAPI staining solution, and observed by ordinary optical microscope and fluorescence microscope. The typical aging characteristics of tumor cells were found: the tumor cells became larger and flattened, the cells shrank, vacuoles formed in the cytoplasm, and sprouting and bubbling occurred. The 尾 -galactosidase (SA- 尾 -gal) staining and the observation of inverted microscope showed that the content of 尾 -galactosidase was increased in the cells of the administration group. The distribution of DNA in the nucleus of the drug treatment group showed the dot aggregation distribution, and with the increase of phycoerythrin concentration, the phenomenon became more and more obvious, which indicated that the proportion of cell senescence was increasing. Then, the effect of phycoerythrin on calcium concentration in tumor cells was detected by Fluo-3/AM staining and confocal laser microscopy. It was found that with the increase of phycoerythrin concentration, phycoerythrin could significantly increase the calcium concentration in tumor cells. There was a dose-dependent relationship. The effects of phycoerythrin on mitochondrial membrane potential in tumor cells were detected by TMRE staining and confocal laser microscopy. It was found that phycoerythrin could significantly decrease mitochondrial membrane potential in tumor cells with the increasing of phycoerythrin concentration. The effect of phycoerythrin on the content of reactive oxygen species (Ros) in tumor cells was detected by laser confocal microscopy (LSCM). The tumor cells were stained with DCFH-DA and the effect of phycoerythrin on the content of reactive oxygen species (Ros) in tumor cells was detected by confocal laser microscopy. It was found that the content of reactive oxygen species in tumor cells increased with the increase of phycoerythrin concentration. On this basis, we preliminarily investigated the pathway of phycoerythrin induced tumor cell senescence. Under confocal laser microscope, the fluorescence intensity of p53, p21 and pRB proteins in human breast cancer MCF-7 cells increased after 48 h treatment with different concentrations of phycoerythrin. With the increase of phycoerythrin concentration, the fluorescence intensity of p53 p21 and pRB protein was also increased, which indicated that phycoerythrin could induce the expression of senescence related proteins p53, p21 and PRB in human breast cancer MCF-7 cells. In conclusion, phycoerythrin has obvious antitumor activity on human breast cancer MCF-7 cells in vitro. By morphological observation, phycoerythrin can induce typical senescence of tumor cells. In terms of mechanism, it may activate the promoter protein -p53 protein of cell senescence by increasing the concentration of calcium ions in tumor cells and increasing the production of reactive oxygen species in tumor cells, thus inducing the occurrence of cell senescence. In conclusion, phycoerythrin has obvious antitumor effect in vitro, and phycoerythrin can induce human breast cancer MCF-7 cell senescence by regulating p53-p21-pRB pathway.
【学位授予单位】：哈尔滨商业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346;R737.9

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