新型人生长激素释放激素类似肽构效关系的研究

发布时间：2017-12-31 18:09

  本文关键词：新型人生长激素释放激素类似肽构效关系的研究　出处：《南方医科大学》2014年硕士论文　论文类型：学位论文

  更多相关文章： 生长激素释放激素 生长激素释放激素类似肽 生物学活性

【摘要】：生长激素释放激素(Growth Hormone-Releasing Hormone, GHRH)是一种下丘脑弓状核和室周区细胞合成及释放的重要内分泌激素,为44个氨基酸组成的活性多肽,其主要功能是调节腺垂体分泌生长激素(Growth Hromone)。GH在许多器官,如肝脏中,刺激释放胰岛素样生长因子(insulin-like growth factor-1和2,IGF-1和2)。同时,GHRH可维持内源性GH和IGF-1的负反馈调节,保持GH和IGF-1在体内的平衡。GHRH、GH和IGFs形成一个神经内分泌轴,对靶细胞起到相应调节作用。人GHRH的N端第一个氨基酸Tyr为其受体配体结合位点,通过与GHRH特异性受体结合,激活第二信使发挥作用。GHRH的N端第二个氨基酸为Gly,人体内的二肽酶能特异性识别此位点,将GHRH水解为没有活性但仍具有免疫特性的多肽,因此,GHRH在人体内的半衰期仅数分钟。随着对GHRH研究的不断深入,研究者们在体内多种生理或病理组织细胞中发现GHRH的存在,亦发现其在体内参与多种重要生理及病理过程,如GHRH有保护心肌梗死后心脏的结构和功能、加速皮肤损伤修复、改善睡眠及认知功能障碍、逆转HIV相关的脂肪代谢异常以及提高糖尿病病人胰岛细胞存活时间及分泌量等功能,拓宽了其在临床应用中的范围。但GHRH存在半衰期短,体外大量制备困难等缺点,限制了GHRH相关研究的深入。GHRH类似肽的研发与进展在很大程度上改善了GHRH上述不足之处,为相关的基础研究及临床应用打下基础。 目的 选择前期研究中构建的肽及其二聚体,测定所有九种肽的释放活性、特异性及受体结合能力,筛选出激素释放特异和活性最高的GHRH类似肽或二聚体。 方法 1.将已转入重组质粒的大肠杆菌BL21(DE3)在LB培养基中37℃300rpm发酵5小时,加入异丙基-β-D-硫代半乳糖苷(ITPG)诱导融合蛋白表达5小时。低温离心机内4℃10000rpm离心5分钟收集大肠杆菌,称重。将收集的表达产物,经裂解细胞、洗涤、乙醇沉淀、酸水解、SP-Sephadex C-25和Sephadex G-25层析柱等方法纯化GHRH;通过高效液相色谱法分析GHRH类似肽纯度,通过质谱法测定GHRH类似肽分子量。 2.将Pro-Pro-hGHRH(1-44)-Gly-Gly-Cys,Pro-hGHRH(1-44)-Gly-Gly-Cys和1Pro-GHRH(2-44)-Gly-Gly-Cys无菌注射用水溶解后放入PH=11.5碱性环境中二聚体化45小时； 3.将所得9中GHRH类似肽及标准肽hGHRH(1-44)-NH2溶解在乳酸盐林格氏液(LRB)中,将S-D雌性大鼠25℃恒温常规饲养后,颈椎脱臼法处死,迅速摘取垂体,用无菌LRB洗净,立即浸入37℃保温的无菌LRB液中进行活性测定。每个垂体放入含有1mL无菌LRB液的小玻璃管(1*10cm)中,37℃水浴保温,每隔10min缓慢振摇一次。1小时后吸取管中液体,立即补加预保温的新LRB液1mL,继续保温1h。吸出的液体待测。垂体总共保温5小时(依次以P1、P2、I3、I4、I5表示连续的5h时间段),前两小时(P1、P2)为空白LRB缓冲液,后三小时(I3、I4、I5)垂体加入相同浓度的各种人GHRH肽-LRB缓冲液。以上均在37℃保温的条件下进行。大鼠GH测定采用Rat Growth Hormone ELISA Kit,以Lowry法测定类似肽浓度。同时,以Rat LH ELISA Kit和Rat ACTH ELISA Kit检测类似肽的特异性。 4. FITC荧光标记活性较高的Pro-Pro-hGHRH(1-44)-Gly-Gly-Cys, Pro-hGHRH(1-44)-Gly-Gly-Cys和1Pro-GHRH(2-44)-Gly-Gly-Cys,在37℃下行离体垂体配体受体结合试验,检验受体配体结合程度； 5.SD大鼠垂体石蜡切片行荧光染色观察受体配体结合情况。 结果 ITPG诱导后,通过特定的酸水解技术和纯化技术可高效率的获得纯度较高的GHRH类似肽。 在活性测试方面,N端Pro-Pro-, Pro-,1Pro-代换,和/或C端Gly-Gly-Cys延伸获得的新型人GHRH类似肽及其二聚体均有一定刺激活性,单体肽中1Pro-GHRH(2-44)-Gly-Gly-Cys肽刺激活性较高；二聚体活性普遍均高于单体肽,其中1Pro-hGHRH(2-44)-Gly-Gly-Cys-Cys-Gly-Gly-hGHRH(2-44)-1Pro活性最高。所有9种肽均有一定刺激特异性。同时,类似肽的活性在一定范围内与浓度成线性关系。并且能够被生长抑素(SST)特异性抑制。 荧光标记的类似肽进行受体配体结合实验,受体配体的结合量在一定范围的浓度内同样成线性关系,与活性测试结果相近。但给予更高浓度的类似肽结合量上升不明显,说明类似肽与受体结合有饱和性。在荧光显微镜下,能够清楚的看到荧光配体与受体在腺垂体细胞膜上广泛均匀结合。 结论 通过对人GHRH进行改构,可获得具有一定与GHRH有相同生物学活性的新类似肽,为未来更深入的基础及临床研究建立基础。
[Abstract]:Growth hormone releasing hormone (Growth Hormone-Releasing, Hormone, GHRH) is an important endocrine hormone and hypothalamic arcuate nucleus cell synthesis and release of the active polypeptide composed of 44 amino acids. Its main function is to regulate pituitary secretion of growth hormone (Growth Hromone).GH in many organs, such as liver, stimulate the release of insulin like growth factor (insulin-like growth and factor-1 2, IGF-1 and 2). At the same time, the negative feedback regulation of GHRH can maintain the endogenous GH and IGF-1, GH and IGF-1 in.GHRH to keep balance in the body, GH and IGFs form a neuroendocrine axis, corresponding to the adjustment function of the target cell. The end of the first N GHRH amino acid Tyr for its receptor ligand binding sites, combined with the specific GHRH receptor, activation of second messenger.GHRH plays a role of N N-terminal second amino acids of Gly, two peptide enzyme in the human body to specific knowledge Don't this site, GHRH was hydrolyzed to peptide, but still has no active immune characteristics so GHRH in the human body has a half-life of only a few minutes. With the deepening research of GHRH, the researchers found that the presence of GHRH in a variety of physiological or pathological tissues, also found in the body is involved in many important physiological and the pathological process, such as GHRH have the structure and function of the heart protection after myocardial infarction, accelerate skin repair, improve sleep and cognitive dysfunction, reverse HIV related to lipid metabolism abnormalities and improve patients' survival time of islet cells and the secretion of other functions, broaden its clinical application scope. But the existence of GHRH half-life in short, shortcomings in vitro preparation difficult, limiting the development and progress of deep.GHRH like peptide GHRH related research to improve the shortcomings of GHRH to a great extent, is related to Basic research and clinical application lay the foundation.
objective
The peptides and their two polymers from the previous studies were selected to determine the release activity, specificity and receptor binding ability of all nine peptides. The GHRH like peptide or two polymer with the highest hormone release and activity was screened out.
Method
Escherichia coli BL21 1. has been transferred to the recombinant plasmid (DE3) in LB medium 300rpm 37 degrees of fermentation for 5 hours, adding isopropyl thiogalactoside beta -D- (ITPG) fusion protein was induced for 5 hours. Within 4 DEG 10000rpm low temperature centrifuge centrifuge for 5 minutes to collect the Escherichia coli expression product will collect the weighing. After cell lysis, washing, ethanol precipitation, acid hydrolysis, purification of GHRH SP-Sephadex C-25 and Sephadex G-25 chromatography method; through HPLC analysis of GHRH like peptide purity, determination of GHRH like peptide molecular weight by mass spectrometry.
2., Pro-Pro-hGHRH (1-44) -Gly-Gly-Cys, Pro-hGHRH (1-44) -Gly-Gly-Cys and 1Pro-GHRH (2-44) -Gly-Gly-Cys were dissolved in sterile water for injection, then placed in PH=11.5 alkaline environment for two polymers for 45 hours.
3.灏嗘墍寰，

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