發(fā)布時間：2017-12-31 18:09

  本文關(guān)鍵詞：新型人生長激素釋放激素類似肽構(gòu)效關(guān)系的研究　出處：《南方醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 生長激素釋放激素 生長激素釋放激素類似肽 生物學(xué)活性

【摘要】：生長激素釋放激素(Growth Hormone-Releasing Hormone, GHRH)是一種下丘腦弓狀核和室周區(qū)細(xì)胞合成及釋放的重要內(nèi)分泌激素,為44個氨基酸組成的活性多肽,其主要功能是調(diào)節(jié)腺垂體分泌生長激素(Growth Hromone)。GH在許多器官,如肝臟中,刺激釋放胰島素樣生長因子(insulin-like growth factor-1和2,IGF-1和2)。同時,GHRH可維持內(nèi)源性GH和IGF-1的負(fù)反饋調(diào)節(jié),保持GH和IGF-1在體內(nèi)的平衡。GHRH、GH和IGFs形成一個神經(jīng)內(nèi)分泌軸,對靶細(xì)胞起到相應(yīng)調(diào)節(jié)作用。人GHRH的N端第一個氨基酸Tyr為其受體配體結(jié)合位點(diǎn),通過與GHRH特異性受體結(jié)合,激活第二信使發(fā)揮作用。GHRH的N端第二個氨基酸為Gly,人體內(nèi)的二肽酶能特異性識別此位點(diǎn),將GHRH水解為沒有活性但仍具有免疫特性的多肽,因此,GHRH在人體內(nèi)的半衰期僅數(shù)分鐘。隨著對GHRH研究的不斷深入,研究者們在體內(nèi)多種生理或病理組織細(xì)胞中發(fā)現(xiàn)GHRH的存在,亦發(fā)現(xiàn)其在體內(nèi)參與多種重要生理及病理過程,如GHRH有保護(hù)心肌梗死后心臟的結(jié)構(gòu)和功能、加速皮膚損傷修復(fù)、改善睡眠及認(rèn)知功能障礙、逆轉(zhuǎn)HIV相關(guān)的脂肪代謝異常以及提高糖尿病病人胰島細(xì)胞存活時間及分泌量等功能,拓寬了其在臨床應(yīng)用中的范圍。但GHRH存在半衰期短,體外大量制備困難等缺點(diǎn),限制了GHRH相關(guān)研究的深入。GHRH類似肽的研發(fā)與進(jìn)展在很大程度上改善了GHRH上述不足之處,為相關(guān)的基礎(chǔ)研究及臨床應(yīng)用打下基礎(chǔ)。 目的 選擇前期研究中構(gòu)建的肽及其二聚體,測定所有九種肽的釋放活性、特異性及受體結(jié)合能力,篩選出激素釋放特異和活性最高的GHRH類似肽或二聚體。 方法 1.將已轉(zhuǎn)入重組質(zhì)粒的大腸桿菌BL21(DE3)在LB培養(yǎng)基中37℃300rpm發(fā)酵5小時,加入異丙基-β-D-硫代半乳糖苷(ITPG)誘導(dǎo)融合蛋白表達(dá)5小時。低溫離心機(jī)內(nèi)4℃10000rpm離心5分鐘收集大腸桿菌,稱重。將收集的表達(dá)產(chǎn)物,經(jīng)裂解細(xì)胞、洗滌、乙醇沉淀、酸水解、SP-Sephadex C-25和Sephadex G-25層析柱等方法純化GHRH;通過高效液相色譜法分析GHRH類似肽純度,通過質(zhì)譜法測定GHRH類似肽分子量。 2.將Pro-Pro-hGHRH(1-44)-Gly-Gly-Cys,Pro-hGHRH(1-44)-Gly-Gly-Cys和1Pro-GHRH(2-44)-Gly-Gly-Cys無菌注射用水溶解后放入PH=11.5堿性環(huán)境中二聚體化45小時； 3.將所得9中GHRH類似肽及標(biāo)準(zhǔn)肽hGHRH(1-44)-NH2溶解在乳酸鹽林格氏液(LRB)中,將S-D雌性大鼠25℃恒溫常規(guī)飼養(yǎng)后,頸椎脫臼法處死,迅速摘取垂體,用無菌LRB洗凈,立即浸入37℃保溫的無菌LRB液中進(jìn)行活性測定。每個垂體放入含有1mL無菌LRB液的小玻璃管(1*10cm)中,37℃水浴保溫,每隔10min緩慢振搖一次。1小時后吸取管中液體,立即補(bǔ)加預(yù)保溫的新LRB液1mL,繼續(xù)保溫1h。吸出的液體待測。垂體總共保溫5小時(依次以P1、P2、I3、I4、I5表示連續(xù)的5h時間段),前兩小時(P1、P2)為空白LRB緩沖液,后三小時(I3、I4、I5)垂體加入相同濃度的各種人GHRH肽-LRB緩沖液。以上均在37℃保溫的條件下進(jìn)行。大鼠GH測定采用Rat Growth Hormone ELISA Kit,以Lowry法測定類似肽濃度。同時,以Rat LH ELISA Kit和Rat ACTH ELISA Kit檢測類似肽的特異性。 4. FITC熒光標(biāo)記活性較高的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℃下行離體垂體配體受體結(jié)合試驗(yàn),檢驗(yàn)受體配體結(jié)合程度； 5.SD大鼠垂體石蠟切片行熒光染色觀察受體配體結(jié)合情況。 結(jié)果 ITPG誘導(dǎo)后,通過特定的酸水解技術(shù)和純化技術(shù)可高效率的獲得純度較高的GHRH類似肽。 在活性測試方面,N端Pro-Pro-, Pro-,1Pro-代換,和/或C端Gly-Gly-Cys延伸獲得的新型人GHRH類似肽及其二聚體均有一定刺激活性,單體肽中1Pro-GHRH(2-44)-Gly-Gly-Cys肽刺激活性較高；二聚體活性普遍均高于單體肽,其中1Pro-hGHRH(2-44)-Gly-Gly-Cys-Cys-Gly-Gly-hGHRH(2-44)-1Pro活性最高。所有9種肽均有一定刺激特異性。同時,類似肽的活性在一定范圍內(nèi)與濃度成線性關(guān)系。并且能夠被生長抑素(SST)特異性抑制。 熒光標(biāo)記的類似肽進(jìn)行受體配體結(jié)合實(shí)驗(yàn),受體配體的結(jié)合量在一定范圍的濃度內(nèi)同樣成線性關(guān)系,與活性測試結(jié)果相近。但給予更高濃度的類似肽結(jié)合量上升不明顯,說明類似肽與受體結(jié)合有飽和性。在熒光顯微鏡下,能夠清楚的看到熒光配體與受體在腺垂體細(xì)胞膜上廣泛均勻結(jié)合。 結(jié)論 通過對人GHRH進(jìn)行改構(gòu),可獲得具有一定與GHRH有相同生物學(xué)活性的新類似肽,為未來更深入的基礎(chǔ)及臨床研究建立基礎(chǔ)。
[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.灝嗘墍寰，

本文編號：1360872