【作者】 付欣；

【导师】 尚德静；

【作者基本信息】 辽宁师范大学 ， 遗传学， 2011， 硕士

【摘要】 硒是一种人体必需的微量元素,许多疾病比如癌症、心血管病等都与体内缺硒有关。在硒的诸多生物功能中,硒的抗癌作用一直被人们广泛关注。硒化合物的存在形式包括有机硒和无机硒两种,有机硒主要包括硒多糖、硒蛋白等,无机硒主要包括硒酸和亚硒酸。尽管无机硒化合物被认为是有效的化学防癌药物,但是有机硒因其低毒性而倍受关注。灵芝硒多糖作为一种有机硒化合物,兼具硒和多糖的功能,并且其生理功能普遍高于硒和多糖,更利于机体吸收。本论文以实验室前期通过灵芝加硒培养,分离纯化后得到的具有抗癌活性组分灵芝硒多糖SeGLP-2B-1为实验材料。将灵芝硒多糖SeGLP-2B-1作用于乳腺癌MCF-7细胞,作用浓度分别为0.0225μM、0.045μM、0.09μM、0.18μM、0.27μM、0.36μM、0.45μM、0.54μM、0.64μM,作用时间为1-72h,并且设置阴性对照组,通过MTT法测定其对细胞生长的抑制作用。得到在48h、72h的半数抑制浓度(IC50)分别为0.264μM、0.246μM。使用DCFH-DA荧光探针测定0.27μM的灵芝硒多糖诱导乳腺癌MCF-7细胞凋亡的过程中,1-72h内细胞活性氧含量变化,同时测定细胞内脂质过氧化物丙二醛(MDA)变化。为了探索活性氧在细胞凋亡中的影响,我们测定了0.27μM灵芝硒多糖诱导细胞凋亡时,1h、3h、6h、8h、24h、48h、72h这7个时间点细胞内的主要抗氧化酶CAT、SOD、GSH-Px的含量变化,进一步研究硒多糖诱导细胞凋亡作用中的氧化应激机制。实验结果表明,灵芝硒多糖SeGLP-2B-1对乳腺癌细胞MCF-7的生长具有抑制作用,随着灵芝硒多糖作用时间的增加,其对细胞生长的抑制率增强,并且这种抑制效果也和作用浓度呈正相关。采用DCFH-DA荧光探针标记,检测到随着灵芝硒多糖作用时间的增加,硒的抗氧化作用增强,细胞内活性氧ROS含量降低,在72h检测到ROS含量到达最低值。并且对作为细胞内活性氧水平的另一个指标的氧化应激脂质过氧化分解产物丙二醛(MDA)ELISA法检测,得到的MDA结果与ROS结果一致。此外,灵芝硒多糖作用乳腺癌细胞MCF-7后,细胞内抗氧化酶SOD,CAT,GSH-Px含量随着作用时间延长逐渐增加。这从一定侧面证明了硒的抗氧化能力,并且推测灵芝硒多糖的抗氧化效应是通过增强细胞内抗氧化酶的活性来实现的。更多还原

【Abstract】 Selenium (Se) is an essential trace element for human being. Se deficiency induces many diseases, such as cancer, cardiovascular disease. In many biological functions of Se, the anticarcinogenic properties of selenium has been widespread concerned. The chemical forms of Se include organic and inorganic Se. The organic Se compounds include Se-containing polysaccharides, Se-containing proteins etc., and the inorganic Se compounds include selenonic acid and the selenite salt. Although inorganic Se compounds are considered as effective anti-cancer drugs, organic Se are being pay highly attention for its higher bioavailability and is safer than inorganic Se species. As a kind of organic Se, Se-containing Polysaccharide from Se-enriched Ganoderma lucidum concurrently has the function of Se and polysaccharide, and therefore, it is crucial to develop suitable organic Se dietary sources for humans.After co-cultivation of ganoderma with selenium, we detached and purified an Se-containing Polysaccharide SeGLP-2B-1 with anticancer activity. The first purpose of present study is to determine the cytotoxic effects of Se-containing Polysaccharide SeGLP-2B–1, at various concentrations (0.0225μM、0.045μM、0.09μM、0.18μM、0.27μM、0.36μM、0.45μM、0.54μM、0.64μM ) and different time-courses (24h、48h、72h respectively), against breast cancer cell line MCF-7. The results of MTT assay showed that the Se-containing Polysaccharide SeGLP-2B-1 inhibited the proliferation of breast cancer cells MCF-7 in a time-dependent and dose-dependent manner. The half inhibitory concentration (IC50) of SeGLP-2B–1 were 0.264 and 0.246μM in the time of 48h and 72h. Then, using DCFH-DA fluorescent probing and malondialdehyde (MDA) assay, the concentrations of intracellular ROS and MDA were detected to explore the possible involvment of ROS in SeGLP-2B-1 induced MCF-7 cell apoptosis.Experiment results show that with Se-containing Polysaccharide time-dependently enhanced selenium anti-oxidation, reduced content of ROS ( 72h detected ROS levels reached the lowest) and MDA.Se-containing Polysaccharide, SeGLP-2B-1, time-dependently increased the contant of antioxidant enzymes (SOD, CAT, GSH-Px) in MCF-7 cells. This partially proved the anti-oxidant ability of selenium, and speculated that the antioxidant effect of Se-containing Polysaccharide might be medited by enhanceing the activity of endogenous anti-oxidase in cells.更多还原