钒配合物抑制PTP1B影响细胞磷酸化的初步研究

发布时间：2018-08-28 18:59

【摘要】：蛋白酪氨酸磷酸酶(PTPs)在细胞信号转导和调节过程中发挥重要作用,广泛影响细胞各种代谢过程如细胞增殖、分化、迁移、凋亡、免疫应答等过程。PTPs的异常表达与许多疾病诸如糖尿病、癌症、肥胖症、类风湿、免疫功能紊乱等密切有关。PTP1B是一种胞内蛋白质,是最早被发现和纯化的蛋白酪氨酸磷酸酶。研究表明,PTP1B在胰岛素受体以及瘦素受体信号通路中起着负调控作用。敲除PTP1B基因的小鼠对胰岛素的敏感性以及高脂肪饮食下对肥胖症的耐受性都得到了很大的提高。因此,PTP1B成为糖尿病新药设计的理想靶标,近年来,其抑制剂的研究受到人们广泛的关注。钒化合物具有胰岛素样活性并能强烈抑制PTPs,PTP1B选择性钒配合物可望发展成为低毒高效的抗糖尿病药物。我们前期研究显示氧钒配合物[VIVO(nbbb)(H20)2](1)和[VIVO(bpmp)2](2)对PTP1B表现较好的选择性,本文在此基础上对其影响细胞磷酸化进行了初步研究,主要工作如下。本文主要工作是运用蛋白免疫印迹法检测氧钒配合物(1)和(2)对三种肿瘤细胞(MCF-7、HepG-2和Hela)内PTPs底物的磷酸化水平的影响进行探索。结果表明：氧钒配合物(1)能明显增强PTP1B作用底物P-Src(Y529). (IR/IGF1R)(PYPYPY115811621163)以及P-EGFR(Y1092)磷酸化,而对TCPTP作用底物P-Src(Y418)磷酸化的影响很小,特别是在MCF-7细胞中,且该配合物提高PTP1B作用底物磷酸化的能力要明显强于阳性对照VOS04,表明这两种钒配合物特别是(1)进入细胞仍然保持对PTP1B的选择性,而且不同的钒配合物对不同细胞的磷酸化影响不同,同一种配合物对不同细胞的作用也不同,为我们今后进一步筛选钒配合物抗糖尿病药物提供了有益的信息。此外,我们还研究了生物小分子钒配合物对蛋白酪氨酸磷酸酶的抑制作用和选择性,结果显示,大部分生物小分子氧钒配合物对PTP1B表现出强烈的抑制作用,化合物[VO(Phe)2]表现出最强的抑制作用,而[VO(Arg)2]、[VO(Oxalate)]、 [VO(Nitrilotriacetate)]和[VO(Citrate)]则呈现较弱的抑制。选择性研究显示,对PTP1B抑制作用较强的配合物对其它酶的抑制作用也较强,而对PTP1B抑制作用较弱的配合物对其它酶的抑制作用相对也较弱,但选择性却有不同。
[Abstract]:Protein tyrosine phosphatase (PTPs) plays an important role in the signal transduction and regulation of cells. It affects many metabolic processes such as cell proliferation, differentiation, migration and apoptosis. The abnormal expression of PTPs in immune response is closely related to many diseases such as diabetes, cancer, obesity, rheumatoid, immune dysfunction and so on. PTP1B is an intracellular protein and is the first protein tyrosine phosphatase found and purified. PTP1B plays a negative role in insulin receptor and leptin receptor signaling pathway. Insulin sensitivity in PTP1B knockout mice and tolerance to obesity in high fat diets were significantly improved. Therefore, PTP1B has become an ideal target for the design of new drugs for diabetes mellitus. In recent years, the research of PTP1B inhibitors has been paid more and more attention. Vanadium compounds with insulin-like activity and strong inhibition of PTPs,PTP1B selective vanadium complexes are expected to develop into low toxicity and high efficiency antidiabetic drugs. Our previous studies showed that [VIVO (nbbb) (H20] 2] (1) and [VIVO (bpmp) 2] (2) exhibited better selectivity to PTP1B. On the basis of these studies, the effects of vanadium oxide complexes [VIVO (nbbb) (H20] 2] (1) and [VIVO (bpmp) 2] (2) on cell phosphorylation were studied. The main work is as follows. In this paper, the effects of vanadium complexes (1) and (2) on the phosphorylation of PTPs substrates in three kinds of tumor cells (MCF-7,HepG-2 and Hela) were investigated by Western blot. The results show that vanadium oxide complex (1) can obviously enhance the substrate P-Src (Y529) of PTP1B. (IR/IGF1R) (PYPYPY115811621163) and P-EGFR (Y1092) phosphorylation, but had little effect on TCPTP substrate P-Src (Y418) phosphorylation, especially in MCF-7 cells. Moreover, the ability of the complexes to increase the phosphorylation of PTP1B substrates was significantly stronger than that of the positive control VOS04,. The results showed that the two vanadium complexes, especially (1) the two vanadium complexes remained selective to PTP1B when they entered the cells. Moreover, different vanadium complexes have different effects on phosphorylation in different cells, and the same complex has different effects on different cells, which provides useful information for the further screening of vanadium complexes for antidiabetic drugs. In addition, we also studied the inhibition and selectivity of small molecular vanadium complexes on protein tyrosine phosphatase. The results showed that most of the small molecular vanadium complexes exhibited strong inhibition on PTP1B. The compound [VO (Phe) _ 2] showed the strongest inhibitory effect, while [VO (Arg) _ 2], [VO (Oxalate)], [VO (Nitrilotriacetate)] and [VO (Citrate)] showed weaker inhibition. The selective studies showed that the complexes with strong inhibitory effect on PTP1B also had stronger inhibition on other enzymes, while the complexes with weaker inhibitory effect on PTP1B had relatively weak inhibition on other enzymes, but the selectivity was different.
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96;O641.4

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