【作者】 郑克岩；

【导师】 罗贵民；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 硒为生物必需微量元素,在人类的生长发育过程中发挥重要的生理功能。生物体内,硒主要以三种形式存在:硒蛋白、硒多糖和硒核酸。研究表明,硒蛋白和硒多糖具有抗氧化、抗肿瘤、提高机体免疫力等生物学功能,对它们结构和作用机制的研究,可以为人类在医学保健上提供有用的资料,为人类的健康成长谋福。硒在人体内最重要的生理功能就是针对活性氧及其衍生物的抗氧化活性,从而构成人体的抗氧化防御体系。在哺乳动物体内,最重要的功能硒蛋白就是谷胱甘肽过氧化物酶(Glutathione peroxidase,GPX),它的主要功能是清除机体在新陈代谢过程中产生的氧自由基,保护细胞的膜结构,抗氧化损伤。晶体结构研究证明,硒以硒代半胱氨酸的形式定位在GPX的活性位点上,是GPX的活性基团,对它的酶活性起关键性的作用。硒多糖主要存在于一些植物和真菌体内,是人体最重要的硒补充剂,进一步研究发现,硒多糖还具有一些其它的功能,例如清除有害的金属离子、提高机体免疫力等。因此,对这两种有机硒化合物进行研究和开发,就成为科学工作者的研究热点。GPX在人体内具有重要的生理活性,但是它的不稳定性和资源局限性导致它的利用率很低,因此,开发具有高GPX活力的人工模拟物就成为研究的重点。人工模拟酶的关键是构建具有与天然酶相似的底物结合部位和活性中心结构。在以前化学合成的小分子模拟物的研究中发现,从仅考虑活性基团而不考虑底物结合位点到二者全部考虑,模拟物的GPX活力有大幅的提高,但是活力均小于天然酶,限制了它们的应用。我们利用天然酶为材料,通过化学修饰其活性位点的丝氨酸残基,得到了接近天然酶活力的GPX模拟物,提示我们这是一种理想的模拟方法。文献表明,谷胱甘肽硫转移酶(glutathione transferase,GST)与GPX具有共同的GSH结合位点,如果在它的活性中心上含有丝氨酸残基(Ser),我们利用化学修饰的方法将其转变为硒代半胱氨酸(Sec),则可能获得高GPX活力的模拟酶。通过对天然酶进行筛选,我们利用人zeta族谷胱甘肽硫转移酶(hGST Z1-1)作为模拟的蛋白骨架。hGST Z1-1的活性中心结构同GPX相似,并且其活性中心的开口处具有两个Ser残基,利于化学修饰和与底物结合,因此是一个理想的模拟材料。化学修饰后得到的Se-hGST Z1-1具有很高的GPX活性,是某些天然酶活力的1.5倍。结果表明进行酶模拟的关键是构建与天然酶相似的活性部位的微环境,这为我们将来的工作提供了理论依据。在开发小分子GPX模拟物的时候,我们惊喜地发现某些模拟物还具有其它的生理活性,其中硒化环糊精对肿瘤细胞的双重作用提示我们,它可以预防和治疗某些癌症,具有潜在的药用价值。2-硒桥联-β-环糊精(2-selenium-bridgedβ-cyclodextrin,2-SeCD)在低剂量时可以促进HeLa细胞的生长,2-SeCD起抗氧化保护细胞的作用;在高剂量时,引起HeLa细胞凋亡,2-SeCD此时为细胞内的GSH耗竭剂,细胞内GSH快速大量的消耗引起细胞内氧化还原态的变化,激活caspase-3,直接引起肿瘤细胞凋亡。2-SeCD的这种双重调节作用是因为其具有谷胱甘肽过氧化物酶、脱氢抗坏血酸还原酶、巯醇转移酶活性,在行使这些功能时,将大量的GSH转化成GSSG,改变了细胞内的氧化还原平衡导致细胞凋亡。硒多糖作为硒的一种功能性化合物由于其在自然界中含量很低,因此对它的化学合成及抗肿瘤活性研究就成为科学家的研究重点。我们利用研究GPX模拟物的化学修饰方法,将蛋白多糖的羟基用PMSF活化,然后用-SeH亲核取代活化的-OH,将硒连接到蛋白多糖分子上,得到具有抗氧化活性的硒化蛋白多糖。生物活性实验研究表明,它具有直接杀死肿瘤细胞的功能,并且还保留了原多糖的免疫增强作用。离体实验表明它的抗肿瘤活性是通过调整细胞内bcl-2和bax的比值来启动肿瘤细胞凋亡。更多还原

【Abstract】 Selenium is an essential trace element in human body. It plays an important role in the human’s metabolism. The deficiency and excessiveness of selenium will lead to the incidence of various diseases. The existent forms of selenium in human are selenoprotein, selenopolysaccharide and selenonucleic acid. Selenium displays its physiological function by means of selenoenzyme. Glutathione peroxidase is one of important selenoenzyme in human body, and plays an important role in the antioxidative defense system of organisms. It can catalyze the reduction of various peroxides by glutathione. It can block the ROS second order reactions induced by lipid peroxides and decrease the damage of body induced by peroxides. It can protect cell membrane and hold out the normal physiological function of cell. Selenopolysaccharide is a perfect selenium supplement and possesses the functions of antioxidation, improving the organism immunity and removal of harmful heavy metal, and so on. The contents of the two organic selenium compounds are very low in nature and affect their application in medical treatment and health protection. A large number of stable mimics could be obtained by chemical modification. Therefore, it becomes the study focus for researchers to mimic natural enzyme by chemical modification.GPX mimics have been studied for a long time, because the early studies only considered mimicking the active group of GPX and did not consider the effect of interaction between mimics and substrate, the activities of mimics were generally rather low. For example, the activity of Ebselen is only 0.99U/μmol. With the development of research, the importance of interaction between mimics and substrate is attracting more and more attention from researchers. A series of GPX mimics with GSH binding site were produced now. Because the poor conformation blocks the approach of GSH to the active site and and reduces the specifically binding ability, the mimics have low activities. Therefore, it become an ideal strategy to obtain the mimics which has the similar activities with natural GPX using chemical mutation of the Ser to Sec in the active site of protein scaffold which has GSH binding site. By this way, we have successful obtained a new mimic which activities are same order of magnitude as natural GPX.The early research of selenopolysaccharide focused on that it is a perfect selenium supplement, but with the development of research, it was found that selenopolysaccharide has synergism between selenium and polysaccharide. Specially significant is its antitumor activity. Due to the lower content in nature, people often use the method enrichment of selenium in plant to obtain a large of selenopolysaccharide. However, the method has long cycle and output instability, a number of resources were wasted. A large of mimics can be obtained by chemical methods. Therefore, the chemical modification becomes an ideal method for obtaining more selenopolysaccharide. After -OH of the polysaccharide were mutated to -SeH, a large of selenopolysaccharide will be harvested.In studying bioactivities of the two organic selenium compounds, we found that they all have antitumor functions. So we studied the antitumor activity of 2-SeCD and selenopolysaccharide in our lab, and further explore their mechanism in the course of antitumor. The data will provide valuable information for its application in clinical medicine.1. Chemical modification of glutathione transferase to imitate GPXThe key to construct mimics with high GPX activities is that the mimic must have GSH binding site and the Sec in the active site must be close to GSH. This means the microenvironment of the active center of mimic should be similar with that of the natural enzymes. According to this principle, we chose hGST Z1-1 as protein scaffold of chemical modification. The GSH binding site of hGST Z1-1 is similar with that of the natural GPX. The active site of hGSTZ1-1 is located in a deep crevice between the N- and C- terminal domains, nestling into a hydrophobic pocket. The characteristic motif of hGSTZ1-1, SSC (Ser14, Ser15, Cys16), locates in the N-terminal region and lines the port of the hydrophobic pocket. The location of both Ser is benefited to change to Sec by chemical mutation and is easy to approach GSH. Without the steric hindrance effect of structure, both the mutated Sec can easy to approach and bind GSH. We have successfully harvested Se-hGSTZ1-1 with high GPX activity by chemical modification, its activity is 8602.3U/μmol and is higher than that of some native GPX. And its activity is also higher than (1.5 fold) that of rabbit liver GPX. Catalytic kinetic analysis of Se-hGSTZ1-1 shows that the double reciprocal plots of the initial velocity versus the concentration of substrates were a family of parallel lines, consistent with a Ping-Pong mechanism similar with that of native GPX.2. Study of the antitumor activity of 2-SeCD and its action mechanism2-selenium-bridgedβ-cyclodextrin (2-SeCD) is a glutathione peroxidase mimic synthesized in our lab previously. It possesses potent antioxidant ability by catalyzing the reduction of hydroperoxides with glutathione and its GPX activity is 7.4 U/μmol that is 7.6 folds of that of Ebselen. In the study of its biological activities, we found low dose of 2-SeCD could inhibit the UV-B induced cell injury and inhibit cell apoptosis, but the effects of high dose of 2-SeCD to cell has not been reported. Here, we have studied the effects of high and low dose 2-SeCD on human cervical cancer cells (HeLa cells) death and proliferation. We found low dose of 2-SeCD could inhibit HeLa cell apoptosis. The antiapoptotic mechanism may be its antioxidant ability and it can protect cells against oxidative damage. High dose of 2-SeCD can induce HeLa cells apoptosis. The apoptotic mechanism may be that it can largely and rapidly deplete GSH, activate caspase-3 and induce the HeLa cell apoptosis, directly. To investigate the reason for decrease of the intracellular GSH, we studied the other biological activity of 2-SeCD, we found it has potential dehydroascorbic acid reductase- and thioltransferase-like activities. In the course of catalysis, 2-SeCD is reduced by GSH, generated CD-SeH and GSSG in the preincubation process and CD-SeH is involved in main catalytic reaction. In a whole catalytic cycle, 1 mole of 2-SeCD can deplete 4 moles of GSH. When the dose of 2-SeCD is very high, the consumption of GSH increases, the content of GSH cannot meet the needs for tumor cell to grow, leading to the change of redox state in tumor cell, activating caspase-3 and inducing the HeLa cell apoptosis. The results show the dual effect of 2-SeCD on HeLa cells growth, which has great potential in the tumor treatment.3. Preparation of selenoproteoglycan and its antitumor activitySelenopolysaccharide has the biological activity of both selenium and polysaccharide, simultaneously. The biological activities of selenopolysaccharides are better than polysaccharides. For example, it can enhance the immunity and antitumor. In nature, the content of selenopolysaccharide is very low and is far from meeting the demands in human medical treatment and health protection. Therefore, it becomes the focus for researchers to artificially synthesize selenopolysaccharide mimic. We gained a selenoproteoglycan (Se-GTP) with GPX activity by converting the–OH of Ganoderma tsugae. proteoglycan (GTP) to–SeH. The chemical modification method was used to study the GPX mimics in our lab. We also studied the antitumor activities of GTP and Se-GTP. Antitumor experiment in vivo showed that the average tumor inhibition percentage is 74.83%. The study of immunocompetence showed the antitumor activity of GTP is correlative with its immunocompetence and related with many factors. Antitumor experiment of GTP and Se-GTP in vivo showed the ability for GTP to kill tumor is very low, but the ability for Se-GTP to kill tumor is better than GTP. The cell death percentages are 86.68% and 15.79%, respectively, when the concentration of Se-GTP and GTP is at 500μg/ml.Se-GTP can induce the increase of Bax expression and decrease of Bcl-2 expression, the balance of Bcl-2/Bax is destroyed and induced cell death. Data show that the mechanism of Se-GTP killing tumor cell relates the direct reduction of cell apoptosis. The immunologic activity research indicated that Se-GTP retained the GTP original immunity function, enhanced the NK cell activity and promoted spleen lymphocyte multiplication. These data show that Se-GTP both retain the GTP biology function and increase the new activity, it has excellent potential as an antitumor medicine.更多还原