【摘要】：目的：建立篩選尼克酰胺磷酸核糖轉(zhuǎn)移酶(NAMPT)抑制劑的方法。 方法：以1,4-二馬來酰亞胺基丁烷(1,4-Bismaleimidobutane, BMB)交聯(lián)G355C/D393C雙突變NAMPT,封堵其催化活性中心,以NAMPT野生型及交聯(lián)型NAMPT蛋白的自發(fā)熒光變化(280nm激發(fā),333nm發(fā)射),來檢測化合物與蛋白的結(jié)合,以核磁共振方法體外檢測化合物對NAMPT催化作用的影響,以MTT方法檢測化合物對細(xì)胞活性的影響。 結(jié)果：FK8660.1-1.0μM能夠濃度依賴的抑制野生型NAMPT自發(fā)熒光,但對BMB-NAMPT的自發(fā)熒光沒有影響。迷迭香酸、洋薊素、1,3二咖啡奎寧酸0.5-50μM能夠濃度依賴的抑制野生型NAMPT和BMB-NAMPT的自發(fā)熒光,對兩種蛋白自發(fā)熒光的抑制率無顯著差異。FK866分子比1:1(化合物:NAMPT蛋白)能夠顯著抑制NAMPT的催化作用,但迷迭香酸、洋薊素、1,3二咖啡奎寧酸分子比100:1(化合物:NAMPT蛋白)對NAMPT的催化作用無抑制作用。10-7M FK866時(shí)間依賴的抑制A549細(xì)胞的活性,10-8～10-5M迷迭香酸、洋薊素、1,3二咖啡奎寧酸對A549的細(xì)胞活性無抑制作用,反而在高濃度下對A549細(xì)胞的細(xì)胞活性有一定的增強(qiáng)作用。 結(jié)論：基于NAMPT和BMB-NAMPT蛋白的內(nèi)源性熒光檢測法可以篩選與NAMPT蛋白結(jié)合的化合物,并分析化合物是否結(jié)合在NAMPT的催化活性部位,迷迭香酸、洋薊素、1,3二咖啡奎寧酸可結(jié)合在NAMPT催化活性位點(diǎn)以外的部位。 目的：建立一種篩選尼克酰胺磷酸核糖轉(zhuǎn)移酶(NAMPT)非酶作用阻斷劑的細(xì)胞模型。 方法：通過突變,獲得酶活作用完全消失的NAMPT蛋白,以熒光光譜法檢測無ATP時(shí)NAMPT的催化作用；以Real time-PCR方法檢測IL-6mRNA的表達(dá),觀察各種NAMPT蛋白誘導(dǎo)BV2細(xì)胞和THP-1細(xì)胞IL-6mRNA表達(dá)的作用；獲得穩(wěn)定轉(zhuǎn)染pIL-6-promoter-luc質(zhì)粒的BV2細(xì)胞株,用于IL-6mRNA表達(dá)的高通量篩選。 結(jié)果：H247A、R311D、R392E、K423E突變均能夠顯著減弱JAMPT蛋白的催化作用,其中R392E突變使NAMPT的催化作用完全消失。NAMPT蛋白能夠誘導(dǎo)BV2細(xì)胞和THP-1細(xì)胞表達(dá)IL-6mRNA,其作用與催化作用的強(qiáng)度呈負(fù)相關(guān)。基于IL-6報(bào)告基因的細(xì)胞模型可以用于NAMPT非酶作用的檢測。 結(jié)論：R392E突變的NAMPT蛋白攻擊BV2細(xì)胞或THP-1細(xì)胞,檢測IL-6mRNA的表達(dá)水平,可以用于篩選NAMPT非酶作用阻斷劑,而穩(wěn)定表達(dá)IL-6報(bào)告基因的BV2細(xì)胞可以作為高通量篩選的細(xì)胞模型。
[Abstract]:Objective: to establish a method for screening nicotinamide phosphate ribonucleosyltransferase (NAMPT) inhibitors. Methods: G355C- / D393C double mutation was used to block the catalytic active sites, and the spontaneous fluorescence changes (280nm excited 333nm emission) of NAMPT proteins were used to detect the binding of the compounds to the proteins, and the catalytic activity sites of NAMPT were blocked by the cross-linked G355C / D393C double mutated NAMPTs (14-Bismaleimidobutane (BMB), and the spontaneous fluorescence changes of NAMPT proteins (excited by 333nm emission) were used to detect the binding of the compounds to the proteins. The effects of the compounds on the catalytic activity of NAMPT were detected by nuclear magnetic resonance (NMR) in vitro, and the effects of the compounds on the cell activity were detected by MTT method. Results: FK8660.1-1.0 渭 M could inhibit wild-type NAMPT autofluorescence in a concentration-dependent manner, but had no effect on BMB-NAMPT autofluorescence. Rosemary acid, artichoidin 1, dicoffee quinic acid 0.5-50 渭 M could inhibit the spontaneous fluorescence of wild type NAMPT and BMB-NAMPT in a concentration-dependent manner. There was no significant difference in the inhibition rate of autofluorescence between the two proteins. FK866 molecule could significantly inhibit the catalytic action of Nmpt compared with 1:1, but rosemary acid, The catalytic effect of 100: 1 (compound: NAMPT protein) on NAMPT was not inhibited. 10-7M FK866 inhibited the activity of A549 cells in a time-dependent manner. The activity of A549 cells was not inhibited by A549, but increased at high concentration. Conclusion: the endogenous fluorescence detection method based on NAMPT and BMB-NAMPT protein can be used to screen the compounds bound to NAMPT protein and analyze whether the compounds are bound to the catalytic active site of NAMPT, rosmarinic acid, or not. Artichokes can bind to a site other than the catalytic activity site of NAMPT. Aim: to establish a cell model for screening nonenzymatic blockers of nicotinamide phosphate ribonucleosyltransferase (NAMPT). Methods: the NAMPT protein whose enzyme activity disappeared completely was obtained by mutagenesis. The catalytic effect of NAMPT without ATP was detected by fluorescence spectrometry, the expression of IL-6 mRNA was detected by Real time-PCR method, and the expression of IL-6 mRNA in BV2 cells and THP-1 cells was observed by various NAMPT proteins. BV2 cell lines transfected stably with pIL-6-promoter-luc plasmid were obtained for high throughput screening of IL-6 mRNA expression. Results the mutation of R311DN R392EK423E significantly attenuated the catalytic effect of JAMPT protein. R392E mutation completely disappeared the catalytic effect of NAMPT. NAMPT protein could induce the expression of IL-6mRNAs in BV2 cells and THP-1 cells, which was negatively correlated with the intensity of catalytic action. The cell model based on IL-6 reporter gene can be used to detect the non-enzyme action of NAMPT. Conclusion the expression level of IL-6 mRNA in BV2 cells or THP-1 cells was assayed by the NAMPT protein of 1: R392E mutation, which could be used to screen non-enzymatic blockers of NAPT, while BV2 cells with stable expression of IL-6 reporter gene could be used as a high throughput screening cell model.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R96

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1 韓雪;建立篩選尼克酰胺磷酸核糖轉(zhuǎn)移酶抑制劑的方法[D];浙江大學(xué);2014年

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