【作者】 王迪；

【导师】 王丽萍；

【作者基本信息】 吉林大学 ， 微生物与生化药学， 2008， 硕士

【摘要】 本实验室设计并合成含His的抗坏血酸过氧化物酶肽类模拟物DhHP-6,其清除过氧化物的活性可达3.9x103U·μmol-1(天然微过氧化物酶MP-11的93%),并经体外细胞实验证明其能够抑制细胞凋亡。Exendin-4是胰高血糖素样肽-1的受体激动剂,能够抵抗DPP-Ⅳ酶水解而具有较长的半衰期,是已上市的治疗2型糖尿病的新药。Exenatide为合成的exendin-4。本实验首先从促进细胞增殖、抗凋亡两方面证明DhHP-6体外对胰岛β细胞具有显著的保护作用。然后将DhHP-6和exenatide进行配比之后作用于胰岛β细胞,经初步实验得到最佳配比并进一步发现两种多肽对促进β细胞增殖具有协同作用。本论文为开发更为有效的治疗2型糖尿病的肽类药物提供了有价值的数据。更多还原

【Abstract】 Diabetes mellitus (DM) is a serious disease challenging human health. Because of its increasing incidence these years, World Health Organization (WHO) recognized DM as one of the main chronic human diseases to be prevented. DM is a clinical syndrome due to the beta cells of the pancreas being unable to produce sufficient insulin. The main forms of DM are type 1 and type 2. Type 1 is insulin-dependent DM , but type 2 is insulin-independent DM. The main causes and mechanisms of DM disease are beta cell dysfunction, defects in insulin secretion and apoptosis of beta cells resulting from glucose toxicity, fat toxicity and oxidation damage. Patients with DM have the obvious resistance to insulin. Moreover, both type 1 and type 2 diabetes have obvious genetic heterogeneity and are affected by various environmental factors.Many drugs of T2DM treatment promote insulin secretion from the beta cells of the pancreas. However, this kind of drugs has a great side-effect that long-term treatment will result in insulin over-secretion and thereby loss of beta cell functions.Beta cell functions of T2DM reduce continuingly with increasing diagnosis years.Whatever any therapy is applied, we can not prevent the continuing reduction of beta cell function later. Thus, a new appropriate therapeutic way is required for beta cell protection. Exendin-4 has 53 percentage of homology with human GLP-1 and high affinity to GLP-1 receptor. The effect of its reducing blood sugar is as 5530 times as GLP-1. Since it is protected from the hydrolysis of DPP-IV, its half life in blood plasma is 26 minutes. Because exendin-4 can increase the cytoplasm of beta cell and restore its function, it has the unique advantage of treating T2DM.MP-8, MP-9 and MP-11 from the hydrolysis of cytochrome C (Cyt C) bind to hemachrome covalently and contain a histidine (his) residue in the peptide so that they have effective prevention and cure for various disease due to radical damage. Our research group designed and synthesized an analogue of anti-bad blood acid peroxide enzyme containing his residue, DhHP-6, which has the similar structure of MP from Cyt C. The activity of cleaning peroxides arrives to 3.9x103U·μmol-1 (93% of natural peroxide enzyme, MP-11). Moreover, in vitro experiment showed that it could protect cell from oxidized damage and thereby inhibit cell apoptosis. Thus, DhHP-6 has a potential role and perspective in preventing and curing the disease due to cell function damage.I used insulinoma cell line, RINm-5F to enhance the activity of cell propagation and anti-apoptosis in this work. It showed that DhHP-6 played a critical role in protecting in vitro beta cells.First of all, MTT assays were utilized to measure the ability of enhancing beta cells propagation by DhHP-6. It was shown that compared with control group, the number of beta cells after DhHP-6 treatment was significantly increased. Moreover, the increasing percentage of cell propagation was increased with the increase of DhHP-6 concentration. Therefore, DhHP-6 could enhance beta cell propagation and have more effective role than exenatide(synthetic exendin-4).Secondly, I constructed the model cells to induce the apoptosis of RINm-5F cells by glucose and sodium palmite co-application. MTT assays, flow cytometry and real time-PCR were utilized to test the anti-apoptosis activities of DhHP-6 and exenatide. The results showed that the livability of the model cells was reduced; flow cytometry indicated that the apoptosis peak rate of model cells was increased compared with the control cells; compared to the control cells, the livability of the drug treatment cells was significantly increased and the effect of beta cell protection was increased with the increase of DhHP-6 concentration treatment; compared to the model cells, the apoptosis peak rate of drug treatment cells was reduced to the level of control cells. Thus, DhHP-6 can inhibit the apoptosis induced by glucose and dissociated fatty acids. Moreover, its valid concentration for treatment was lower than exenatide. The resultes of real time-PCR showed that,DhHP-6 and exenatide could preventβ-cell apoptosis induced by FFA and glucose partly through sirt1 and bcl-2 involved Pathway.DhHP-6 and exenatide could promote the RINm-5F cell propagation and inhibit its apoptosis separately, but underlying different mechanism. This work further studied whether these two peptides had the cooperating effect on treating beta cells.DhHP-6 and exenatide were mixed to treat the beta cells. The results showed that when exenatide and DhHP-6 was mixed by 4:1(μM), the mixture had the best effect on promoting beta cell propagation. However, when the mixture concentration was 1:2.5(μM), anti-apoptosis activity was best. It was further shown that these two peptides had the cooperating effect on enhancing the beta cell propagation but not on inhibiting the beta cell apoptosis.The results above indicated that DhHP-6 and exenatide could play a role in protecting beta cells separately. Furthermore, their mixture by certain proportion could also protect beta cells and maybe have cooperating effect on enhangcing beta cells propagation。which could give the contribution to developing the new effective drugs for T2DM.更多还原