【摘要】：蛋白酪氨酸磷酸酶(PTPs)在細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)和調(diào)節(jié)過程中發(fā)揮重要作用,廣泛影響細(xì)胞各種代謝過程如細(xì)胞增殖、分化、遷移、凋亡、免疫應(yīng)答等過程。PTPs的異常表達(dá)與許多疾病諸如糖尿病、癌癥、肥胖癥、類風(fēng)濕、免疫功能紊亂等密切有關(guān)。PTP1B是一種胞內(nèi)蛋白質(zhì),是最早被發(fā)現(xiàn)和純化的蛋白酪氨酸磷酸酶。研究表明,PTP1B在胰島素受體以及瘦素受體信號(hào)通路中起著負(fù)調(diào)控作用。敲除PTP1B基因的小鼠對(duì)胰島素的敏感性以及高脂肪飲食下對(duì)肥胖癥的耐受性都得到了很大的提高。因此,PTP1B成為糖尿病新藥設(shè)計(jì)的理想靶標(biāo),近年來,其抑制劑的研究受到人們廣泛的關(guān)注。釩化合物具有胰島素樣活性并能強(qiáng)烈抑制PTPs,PTP1B選擇性釩配合物可望發(fā)展成為低毒高效的抗糖尿病藥物。我們前期研究顯示氧釩配合物[VIVO(nbbb)(H20)2](1)和[VIVO(bpmp)2](2)對(duì)PTP1B表現(xiàn)較好的選擇性,本文在此基礎(chǔ)上對(duì)其影響細(xì)胞磷酸化進(jìn)行了初步研究,主要工作如下。本文主要工作是運(yùn)用蛋白免疫印跡法檢測氧釩配合物(1)和(2)對(duì)三種腫瘤細(xì)胞(MCF-7、HepG-2和Hela)內(nèi)PTPs底物的磷酸化水平的影響進(jìn)行探索。結(jié)果表明：氧釩配合物(1)能明顯增強(qiáng)PTP1B作用底物P-Src(Y529). (IR/IGF1R)(PYPYPY115811621163)以及P-EGFR(Y1092)磷酸化,而對(duì)TCPTP作用底物P-Src(Y418)磷酸化的影響很小,特別是在MCF-7細(xì)胞中,且該配合物提高PTP1B作用底物磷酸化的能力要明顯強(qiáng)于陽性對(duì)照VOS04,表明這兩種釩配合物特別是(1)進(jìn)入細(xì)胞仍然保持對(duì)PTP1B的選擇性,而且不同的釩配合物對(duì)不同細(xì)胞的磷酸化影響不同,同一種配合物對(duì)不同細(xì)胞的作用也不同,為我們今后進(jìn)一步篩選釩配合物抗糖尿病藥物提供了有益的信息。此外,我們還研究了生物小分子釩配合物對(duì)蛋白酪氨酸磷酸酶的抑制作用和選擇性,結(jié)果顯示,大部分生物小分子氧釩配合物對(duì)PTP1B表現(xiàn)出強(qiáng)烈的抑制作用,化合物[VO(Phe)2]表現(xiàn)出最強(qiáng)的抑制作用,而[VO(Arg)2]、[VO(Oxalate)]、 [VO(Nitrilotriacetate)]和[VO(Citrate)]則呈現(xiàn)較弱的抑制。選擇性研究顯示,對(duì)PTP1B抑制作用較強(qiáng)的配合物對(duì)其它酶的抑制作用也較強(qiáng),而對(duì)PTP1B抑制作用較弱的配合物對(duì)其它酶的抑制作用相對(duì)也較弱,但選擇性卻有不同。
[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.
【學(xué)位授予單位】：山西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R96;O641.4

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