本文關(guān)鍵詞： 銅綠假單胞菌 密度感應(yīng)系統(tǒng) 3OC_(12)-HSL 化學(xué)合成 肥大細(xì)胞 細(xì)胞凋亡 鈣離子 脫顆粒 細(xì)胞因子　出處：《華中科技大學(xué)》2009年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 目的：通過(guò)化學(xué)方法合成銅綠假單胞菌Quorum Sensing(QS)信號(hào)分子3OC_(12)-HSL，并鑒定其結(jié)構(gòu)、純度及其生物活性。觀察不同濃度3OC_(12)-HSL分子對(duì)肥大細(xì)胞細(xì)胞活力、細(xì)胞凋亡及胞內(nèi)鈣離子作用，同時(shí)觀察肥大細(xì)胞釋放IL-4，IL-6，組胺的釋放及脫顆�，F(xiàn)象。 方法：采用以(L)-甲硫氨酸為基礎(chǔ)的合成路線(xiàn)，經(jīng)與碘甲烷成甲基硫摀鹽、水解、環(huán)合得到(S)-高絲氨酸內(nèi)酯鹽酸鹽；(S)-高絲氨酸內(nèi)酯環(huán)與相應(yīng)的酰氯反應(yīng)或與�；醽啴惐サ目s合反應(yīng)制得目的物。合成產(chǎn)物經(jīng)質(zhì)譜、磁共振確定其結(jié)構(gòu)，經(jīng)高效液相色譜確定其純度，并采用帶lacZ為報(bào)告質(zhì)粒的3OC_(12)-HSL分子感應(yīng)菌株E.coli MG4(pKDT17)檢測(cè)其生物活性。通過(guò)化學(xué)合成具生物活性的3OC_(12)-HSL分子，以不同濃度(6．25-100μM)3OC_(12)-HSL分子作用于小鼠肥大細(xì)胞系P815，用WST-1方法觀察不同時(shí)間點(diǎn)細(xì)胞活力改變，以Annexin V／PI雙染法檢測(cè)凋亡效應(yīng)以及通過(guò)共聚焦觀察鈣離子熒光探針Fluo-3 AM方法檢測(cè)胞內(nèi)鈣離子變化。分別用ELISA方法測(cè)定P815釋放IL-4和IL-6情況，同時(shí)檢測(cè)3OC_(12)-HSL分子作用P815和HMC-1細(xì)胞后釋放組胺(ELISA)情況及電鏡觀察脫顆粒現(xiàn)象。 結(jié)果：以(S)-高絲氨酸內(nèi)酯鹽酸鹽計(jì)算，得到3OC_(12)-HSL的收率為37．01％，它們的結(jié)構(gòu)均經(jīng)紫外，質(zhì)譜，核磁等分析確證與天然結(jié)構(gòu)相同，高效液相色譜檢測(cè)3OC_(12)-HSL純度為99．6％。AHLs感應(yīng)菌株E．coli MT102(pJBA132)經(jīng)平板“T”字檢測(cè)顯示16h可見(jiàn)明顯信號(hào)分子分泌，感應(yīng)菌株E.coli MG4(pKDT17)顯示出相似檢測(cè)敏感性，通過(guò)平板檢測(cè)法，人工合成3OC_(12)-HSL分子可誘導(dǎo)感應(yīng)菌株E.coli MG4產(chǎn)生與銅綠假單胞菌相似效果。細(xì)胞活力觀察中，100μM濃度3OC_(12)-HSL分子干預(yù)后的2h至24h，肥大細(xì)胞P815細(xì)胞活力持續(xù)下降；50-100μM作用12h后可明顯抑制P815細(xì)胞活力(P＜0．05)，50μM-100μM作用12h后顯微鏡下觀察可見(jiàn)細(xì)胞明顯皺縮。細(xì)胞凋亡方面，50μM濃度3OC_(12)-HSL作用4h后，超過(guò)20%的P815細(xì)胞出現(xiàn)了凋亡現(xiàn)象，而100μM濃度3OC_(12)-HSL作用后，有超過(guò)40％細(xì)胞出現(xiàn)凋亡。在100μM濃度下，3OC_(12)-HSL分子可引起胞內(nèi)鈣離子濃度的升高，而在50μM濃度下，，鈣離子濃度變化相對(duì)100μM不顯著，但與對(duì)照組和DMSO組相比仍有所升高。促細(xì)胞因子分泌觀察中，經(jīng)6．25-100μM濃度3OC_(12)-HSL分子在與肥大細(xì)胞P815相互作用12和24h后，IL-4的釋放相比對(duì)照組并未產(chǎn)生顯著變化。P815細(xì)胞在經(jīng)6．25-12μM濃度3OC_(12)-HSL分子刺激24h時(shí)，IL-6表達(dá)較對(duì)照組顯著升高(P＜0．05)，在25，50及100μM刺激中，IL-6呈逐漸下降趨勢(shì)(P＜0．05)。而在刺激的第12h時(shí)，變化并不顯著。脫顆粒觀察中，P815細(xì)胞經(jīng)100μM濃度3OC_(12)-HSL分子刺激P815細(xì)胞30min后，并未檢測(cè)出其組胺分泌的差異，但HMC-1細(xì)胞經(jīng)100μM 3OC_(12)-HSL刺激后其組胺釋放較對(duì)照組顯著升高(P＜0．05)。電鏡觀察顯示P815細(xì)胞所含顆粒較HMC-1少，形態(tài)上不具備成熟肥大細(xì)胞的典型特征。HMC-1細(xì)胞經(jīng)3OC_(12)-HSL刺激后可觀察到明顯脫顆�，F(xiàn)象。 結(jié)論：成功合成了具有與天然銅綠假單胞菌QS信號(hào)分子相同結(jié)構(gòu)的3OC_(12)-HSL分子，該分子具備天然生物活性。3OC_(12)-HSL分子可抑制肥大細(xì)胞活力，細(xì)胞凋亡是造成活力抑制的原因之一，而胞內(nèi)鈣離子的升高可能參與了這一過(guò)程。3OC_(12)-HSL分子可刺激小鼠肥大細(xì)胞釋放IL-6，并可使典型肥大細(xì)胞釋放組胺且發(fā)生脫顆�，F(xiàn)象，提示肥大細(xì)胞可能參與了銅綠假單胞菌感染免疫過(guò)程，3OC_(12)-HSL分子在其中發(fā)揮了一定作用。
[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 鏄庢樉鐨辯緝.緇嗚優(yōu)鍑嬩骸鏂歸潰,50渭M嫻撳害3OC_(12)-HSL浣滅敤4h鍚

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