铜绿假单胞菌Quorum Sensing信号分子N-（3-oxododecanoyl）homoserine lacton

发布时间：2018-01-22 16:19

  本文关键词： 铜绿假单胞菌 密度感应系统 3OC_(12)-HSL 化学合成 肥大细胞 细胞凋亡 钙离子 脱颗粒 细胞因子　出处：《华中科技大学》2009年博士论文　论文类型：学位论文

【摘要】： 目的：通过化学方法合成铜绿假单胞菌Quorum Sensing(QS)信号分子3OC_(12)-HSL，并鉴定其结构、纯度及其生物活性。观察不同浓度3OC_(12)-HSL分子对肥大细胞细胞活力、细胞凋亡及胞内钙离子作用，同时观察肥大细胞释放IL-4，IL-6，组胺的释放及脱颗粒现象。 方法：采用以(L)-甲硫氨酸为基础的合成路线，经与碘甲烷成甲基硫摀盐、水解、环合得到(S)-高丝氨酸内酯盐酸盐；(S)-高丝氨酸内酯环与相应的酰氯反应或与酰化丙二酸亚异丙酯的缩合反应制得目的物。合成产物经质谱、磁共振确定其结构，经高效液相色谱确定其纯度，并采用带lacZ为报告质粒的3OC_(12)-HSL分子感应菌株E.coli MG4(pKDT17)检测其生物活性。通过化学合成具生物活性的3OC_(12)-HSL分子，以不同浓度(6．25-100μM)3OC_(12)-HSL分子作用于小鼠肥大细胞系P815，用WST-1方法观察不同时间点细胞活力改变，以Annexin V／PI双染法检测凋亡效应以及通过共聚焦观察钙离子荧光探针Fluo-3 AM方法检测胞内钙离子变化。分别用ELISA方法测定P815释放IL-4和IL-6情况，同时检测3OC_(12)-HSL分子作用P815和HMC-1细胞后释放组胺(ELISA)情况及电镜观察脱颗粒现象。 结果：以(S)-高丝氨酸内酯盐酸盐计算，得到3OC_(12)-HSL的收率为37．01％，它们的结构均经紫外，质谱，核磁等分析确证与天然结构相同，高效液相色谱检测3OC_(12)-HSL纯度为99．6％。AHLs感应菌株E．coli MT102(pJBA132)经平板“T”字检测显示16h可见明显信号分子分泌，感应菌株E.coli MG4(pKDT17)显示出相似检测敏感性，通过平板检测法，人工合成3OC_(12)-HSL分子可诱导感应菌株E.coli MG4产生与铜绿假单胞菌相似效果。细胞活力观察中，100μM浓度3OC_(12)-HSL分子干预后的2h至24h，肥大细胞P815细胞活力持续下降；50-100μM作用12h后可明显抑制P815细胞活力(P＜0．05)，50μM-100μM作用12h后显微镜下观察可见细胞明显皱缩。细胞凋亡方面，50μM浓度3OC_(12)-HSL作用4h后，超过20%的P815细胞出现了凋亡现象，而100μM浓度3OC_(12)-HSL作用后，有超过40％细胞出现凋亡。在100μM浓度下，3OC_(12)-HSL分子可引起胞内钙离子浓度的升高，而在50μM浓度下，，钙离子浓度变化相对100μM不显著，但与对照组和DMSO组相比仍有所升高。促细胞因子分泌观察中，经6．25-100μM浓度3OC_(12)-HSL分子在与肥大细胞P815相互作用12和24h后，IL-4的释放相比对照组并未产生显著变化。P815细胞在经6．25-12μM浓度3OC_(12)-HSL分子刺激24h时，IL-6表达较对照组显著升高(P＜0．05)，在25，50及100μM刺激中，IL-6呈逐渐下降趋势(P＜0．05)。而在刺激的第12h时，变化并不显著。脱颗粒观察中，P815细胞经100μM浓度3OC_(12)-HSL分子刺激P815细胞30min后，并未检测出其组胺分泌的差异，但HMC-1细胞经100μM 3OC_(12)-HSL刺激后其组胺释放较对照组显著升高(P＜0．05)。电镜观察显示P815细胞所含颗粒较HMC-1少，形态上不具备成熟肥大细胞的典型特征。HMC-1细胞经3OC_(12)-HSL刺激后可观察到明显脱颗粒现象。 结论：成功合成了具有与天然铜绿假单胞菌QS信号分子相同结构的3OC_(12)-HSL分子，该分子具备天然生物活性。3OC_(12)-HSL分子可抑制肥大细胞活力，细胞凋亡是造成活力抑制的原因之一，而胞内钙离子的升高可能参与了这一过程。3OC_(12)-HSL分子可刺激小鼠肥大细胞释放IL-6，并可使典型肥大细胞释放组胺且发生脱颗粒现象，提示肥大细胞可能参与了铜绿假单胞菌感染免疫过程，3OC_(12)-HSL分子在其中发挥了一定作用。
[Abstract]:Objective: through the chemical synthesis method of Pseudomonas aeruginosa Quorum Sensing (QS) 3OC_ (12) -HSL signal molecules, and identify its structure, purity and biological activity. The effects of different concentrations of 3OC_ (12) activity of -HSL molecules on mast cell, apoptosis and intracellular calcium ions, and observation of mast cells to release IL-4 IL-6, the release of histamine and degranulation.
Methods: using (L) - based route for the synthesis of methionine, the methyl iodide salt and sulfur cover, hydrolysis, cyclization to get (S) - L-homoserine lactone hydrochloride; (S) - homoserine lactone ring and corresponding chloride reaction or acylation of isopropylidene malonate condensation reaction system purpose. Synthetic products by mass spectrometry, magnetic resonance imaging to determine its structure by HPLC to determine its purity, and with lacZ as the reporter plasmid 3OC_ (12) -HSL molecular induction strain E.coli (pKDT17) MG4 and investigate its biological activity. By chemical synthesis with biological activity of 3OC_ (12) -HSL with different concentrations (6.25-100, M) 3OC_ (12) -HSL molecules in murine mast cell line P815, using WST-1 method to observe the cell viability at different time points change, with Annexin V / PI double staining method to detect apoptosis and observe the calcium ion fluorescence probe Fluo-3 AM detected by confocal The changes of intracellular calcium were measured. ELISA was used to determine the release of IL-4 and IL-6 from P815. Meanwhile, the release of histamine (ELISA) and the degranulation phenomenon under electron microscope were detected by 3OC_ (12) -HSL molecule after P815 and HMC-1 cells.
Results: (S) - L-homoserine lactone hydrochloride, 3OC_ (12) -HSL yield was 37.01% and their structures were confirmed by UV, MS, NMR analysis confirmed the natural and uniform structure, high performance liquid chromatography detection of 3OC_ (12) -HSL purity of 99.6%.AHLs strain E.coli MT102 (pJBA132) induced by the word "T" tablet showed 16h obvious secreted signaling molecules, induction of strain E.coli MG4 (pKDT17) showed similar detection sensitivity by plate assay, synthetic 3OC_ (12) -HSL molecules can induce induction of strain E.coli MG4 and Pseudomonas aeruginosa similar effect. Cell viability was observed in 100 M concentration 3OC_ (12) -HSL molecules after the intervention of 2H to 24h, mast cells, P815 cell activity continued to decline; 50-100 M 12h can significantly inhibit P815 cell viability (P < 0.05), 50 M-100 M after 12h were observed under microscope 鏄庢樉鐨辩缉.缁嗚優鍑嬩骸鏂归潰,50渭M娴撳害3OC_(12)-HSL浣滅敤4h鍚

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