本文選題：tigerinin-1R　切入點：二硫鍵　出處：《吉林大學》2017年碩士論文　論文類型：學位論文

【摘要】：隨著2型糖尿病發(fā)病率的逐年增高,開發(fā)具有促進胰島素釋放能力的新型藥物制劑成為人們研究的熱點。而兩棲動物皮膚分泌物作為天然肽庫也備受關注。Tigerinin-1R(Arg-Val-Cys-Ser-Ala-Ile-Pro-Leu-Pro-Ile-Cys-His-NH2)是從蛙皮分泌物中分離得到的一種含有一對二硫鍵的陽離子多肽。Tigerinin-1R缺少抗菌活性,但是能有效促進胰島素分泌、提高體內(nèi)糖耐量,而且tigerinin-1R對正常血紅細胞具有極低的溶血活性。本研究中,我們以tigerinin-1R作為親本肽,設計了一系列tigerinin-1R類似物,包括tigerinin-cyclic,tigerinin-1R-L4,tigerinin-linear,[C3K]tigerinin-1R以及[C11K]tigerinin-1R來研究其構(gòu)效關系。首先通過Fmoc固相合成法合成所有多肽,利用空氣氧化法氧化含有一對半胱氨酸的多肽成環(huán),通過高效液相色譜和質(zhì)譜對多肽進行純化鑒定。并利用圓二色光譜儀表征多肽二級結(jié)構(gòu),發(fā)現(xiàn)tigerinin-1R系列多肽都呈近α-螺旋結(jié)構(gòu),其中類似物中[C11K]tigerinin-1R與親本肽最為相似。其次,以人血紅細胞和INS-1胰島瘤細胞為對象,研究了tigerinin-1R及其類似物的細胞毒性。溶血實驗證實所有多肽在高達500μM的作用濃度也均不會造成溶血毒性;MTT實驗同樣證實,所有多肽其類似物在高達100μM的作用濃度時也不會對INS-1細胞產(chǎn)生明顯的細胞毒性。進一步,我們對tigerinin-1R及其類似物促進胰島素分泌的影響及其促胰島素釋放的機制進行了研究。ELISA結(jié)果證實,tigerinin-1R在2.8 m M的葡糖糖環(huán)境中都能促進胰島素分泌并且一定范圍內(nèi)呈濃度依賴性。當多肽類似物作用濃度都是10-5 M的時候,它們都顯示出不同的促胰島素釋放活性。其中[C11K]tigerinin-1R展現(xiàn)出最高的促胰島素釋放能力。LDH釋放實驗證明胰島素釋放的增加并不是因為細胞膜遭到破壞。以Fluo3-am作為鈣離子探針,通過激光共聚焦技術,我們證實tigerinin-1R以及[C11K]tigerinin-1R促進胰島素釋放的機制涉及到鈣離子內(nèi)流。利用昆明鼠為動物模型研究對糖耐量的影響,同對照組相比,腹腔注射tigerinin-1R以及[C11K]tigerinin-1R 30 min后血糖濃度明顯下降,其中,[C11K]tigerinin-1R作用組效力低于tigerinin-1R。血清穩(wěn)定性實驗表明[C11K]tigerininin-1R在10%胎牛血清及37℃環(huán)境下穩(wěn)定性遠低于tigerinin-1R。最后,我們還研究了tigerinin-1R系列多肽對INS-1細胞受到損傷的保護能力。MTT實驗表明10-9、10-7、10-5 M的tigerinin-1R系列多肽對棕櫚酸造成的高脂損傷具有保護作用。Hoechst 33258染色實驗也證實10-5 M的tigerinin-1R對高脂損傷具有保護作用。MTT實驗也及進一步證實適宜濃度的tigerinin-1R對H2O2造成的氧化損傷有保護作用。通過本課題的研究,我們證實二硫鍵對tigerinin-1R發(fā)揮促胰島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類似物在細胞水平上表現(xiàn)出比親本肽tigerinin-1R更好的促胰島素釋放活性,它的作用機制涉及到鈣離子內(nèi)流。在動物水平上,[C11K]tigerinin-1R同樣具有降低血糖的能力,但是其穩(wěn)定性低于tigerinin-1R導致其活性偏低。進一步的研究發(fā)現(xiàn),適當濃度的tigerinin-1R系列多肽對受到棕櫚酸誘導產(chǎn)生的高脂損傷和過氧化氫造成的氧化損傷的INS-1細胞保護作用。Tigerinin-1R及其類似物展現(xiàn)出用于2型糖尿病治療的潛力。
[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.

【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R91

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