Tigerinin-1R的构效关系与体内外活性研究

发布时间：2018-03-21 13:44

本文选题：tigerinin-1R　切入点：二硫键　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：随着2型糖尿病发病率的逐年增高,开发具有促进胰岛素释放能力的新型药物制剂成为人们研究的热点。而两栖动物皮肤分泌物作为天然肽库也备受关注。Tigerinin-1R(Arg-Val-Cys-Ser-Ala-Ile-Pro-Leu-Pro-Ile-Cys-His-NH2)是从蛙皮分泌物中分离得到的一种含有一对二硫键的阳离子多肽。Tigerinin-1R缺少抗菌活性,但是能有效促进胰岛素分泌、提高体内糖耐量,而且tigerinin-1R对正常血红细胞具有极低的溶血活性。本研究中,我们以tigerinin-1R作为亲本肽,设计了一系列tigerinin-1R类似物,包括tigerinin-cyclic,tigerinin-1R-L4,tigerinin-linear,[C3K]tigerinin-1R以及[C11K]tigerinin-1R来研究其构效关系。首先通过Fmoc固相合成法合成所有多肽,利用空气氧化法氧化含有一对半胱氨酸的多肽成环,通过高效液相色谱和质谱对多肽进行纯化鉴定。并利用圆二色光谱仪表征多肽二级结构,发现tigerinin-1R系列多肽都呈近α-螺旋结构,其中类似物中[C11K]tigerinin-1R与亲本肽最为相似。其次,以人血红细胞和INS-1胰岛瘤细胞为对象,研究了tigerinin-1R及其类似物的细胞毒性。溶血实验证实所有多肽在高达500μM的作用浓度也均不会造成溶血毒性;MTT实验同样证实,所有多肽其类似物在高达100μM的作用浓度时也不会对INS-1细胞产生明显的细胞毒性。进一步,我们对tigerinin-1R及其类似物促进胰岛素分泌的影响及其促胰岛素释放的机制进行了研究。ELISA结果证实,tigerinin-1R在2.8 m M的葡糖糖环境中都能促进胰岛素分泌并且一定范围内呈浓度依赖性。当多肽类似物作用浓度都是10-5 M的时候,它们都显示出不同的促胰岛素释放活性。其中[C11K]tigerinin-1R展现出最高的促胰岛素释放能力。LDH释放实验证明胰岛素释放的增加并不是因为细胞膜遭到破坏。以Fluo3-am作为钙离子探针,通过激光共聚焦技术,我们证实tigerinin-1R以及[C11K]tigerinin-1R促进胰岛素释放的机制涉及到钙离子内流。利用昆明鼠为动物模型研究对糖耐量的影响,同对照组相比,腹腔注射tigerinin-1R以及[C11K]tigerinin-1R 30 min后血糖浓度明显下降,其中,[C11K]tigerinin-1R作用组效力低于tigerinin-1R。血清稳定性实验表明[C11K]tigerininin-1R在10%胎牛血清及37℃环境下稳定性远低于tigerinin-1R。最后,我们还研究了tigerinin-1R系列多肽对INS-1细胞受到损伤的保护能力。MTT实验表明10-9、10-7、10-5 M的tigerinin-1R系列多肽对棕榈酸造成的高脂损伤具有保护作用。Hoechst 33258染色实验也证实10-5 M的tigerinin-1R对高脂损伤具有保护作用。MTT实验也及进一步证实适宜浓度的tigerinin-1R对H2O2造成的氧化损伤有保护作用。通过本课题的研究,我们证实二硫键对tigerinin-1R发挥促胰岛素分泌作用而言并不是必需的,但其存在是合理的。以Lys取代C端Cys形成的[C11K]tigerinin-1R类似物在细胞水平上表现出比亲本肽tigerinin-1R更好的促胰岛素释放活性,它的作用机制涉及到钙离子内流。在动物水平上,[C11K]tigerinin-1R同样具有降低血糖的能力,但是其稳定性低于tigerinin-1R导致其活性偏低。进一步的研究发现,适当浓度的tigerinin-1R系列多肽对受到棕榈酸诱导产生的高脂损伤和过氧化氢造成的氧化损伤的INS-1细胞保护作用。Tigerinin-1R及其类似物展现出用于2型糖尿病治疗的潜力。
[Abstract]:With the incidence of type 2 diabetes has increased year by year, the development of new drugs to promote insulin release ability becomes the focus of research. The amphibian animal skin secretions as a natural peptide library is also of concern.Tigerinin-1R (Arg-Val-Cys-Ser-Ala-Ile-Pro-Leu-Pro-Ile-Cys-His-NH2) is a kind of two disulfide containing a cationic peptide.Tigerinin-1R lacks antibacterial activity have been isolated from frog skin secretions in, but can effectively promote insulin secretion, improve the tolerance in sugar, and tigerinin-1R of normal red blood cells have very low hemolytic activity. In this study, we take tigerinin-1R as the parent peptide, designed a series of tigerinin-1R analogues, including tigerinin-cyclic, tigerinin-1R-L4, tigerinin-linear, [C3K]tigerinin-1R and [C11K]tigerinin-1R to study. Effect relationship. First through Fmoc solid phase synthesis method All peptides, polypeptides into a ring containing a pair of cysteine by air oxidation, by high performance liquid chromatography and mass spectrometry identification of peptide was purified by circular dichroism spectrometer. And two characterization of polypeptide two level structure, tigerinin-1R series of peptide was nearly alpha helix structure, including [C11K]tigerinin-1R and its parent peptide analogs the most similar. Secondly, with human red blood cells and INS-1 insulinoma cells as the object, the cytotoxicity of tigerinin-1R and its analogues were studied. The hemolysis test confirmed that all peptides in concentration up to 500 M were also not cause hemolytic toxicity; MTT experiment also confirmed that all peptides analogues do not produce cells the toxicity of INS-1 cells was in concentration up to 100 M. Further, we on the tigerinin-1R and its analogues to promote insulin secretion and insulin releasing machine Researched.ELISA confirmed that tigerinin-1R can promote insulin secretion and cells in a dose-dependent manner. In 2.8 m M glucose environment. When the concentration of peptide analogs are 10-5 M, both of which show different insulin releasing activity. The [C11K]tigerinin-1R showed the highest insulin the release ability of.LDH release experiments show that insulin release does not increase because the cell membrane was destroyed. With Fluo3-am as the calcium probe by laser confocal microscopy, we demonstrate that the tigerinin-1R and the [C11K] tigerinin-1R mechanism to promote the release of insulin involves calcium influx. Using Kunming rats as animal model to study effects on glucose tolerance, compared with the control group, intraperitoneal injection of tigerinin-1R and [C11K]tigerinin-1R blood glucose concentration decreased significantly after 30 min of the [C11K] tigerinin-1R The role of group effectiveness than tigerinin-1R. serum stability experiments showed that [C11K]tigerininin-1R in 10% fetal bovine serum and 37 DEG C under the environment of stability is much lower than that of tigerinin-1R.. Finally, we also study the series of tigerinin-1R peptide on INS-1 cell by.MTT experiment showed that the protection ability of injury has a protective effect of.Hoechst 33258 staining experiments also confirmed that 10-5 M tigerinin-1R has a protective effect of.MTT experiment and further confirmed the protective effect of the suitable concentration of tigerinin-1R on oxidative damage caused by H2O2 on high fat and high fat tigerinin-1R damage caused by the M series of polypeptide 10-9,10-7,10-5 on palmitic acid injury. Through this study, we identified two disulfide bonds of tigerinin-1R play a role in promoting insulin secretion is not required, but its existence is reasonable in Lys substituted [C11K]tigerinin-1R analogues C end Cys formation at the cellular level Show than the insulin releasing activity peptide tigerinin-1R better, its mechanism involves calcium influx. In animal level, [C11K]tigerinin-1R also can reduce blood sugar, but its stability is lower than that of tigerinin-1R due to its low activity. Further study found that tigerinin-1R series of polypeptide with proper concentration caused by high lipid damage and hydrogen peroxide by palmitic acid induced oxidative damage in INS-1 cells and the protective effect of.Tigerinin-1R analogues show a potential for the treatment of type 2 diabetes.

【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91

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