本文選題：銅綠假單胞菌 + 苯磺酸氨氯地平�。� 參考：《福建醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的 群體感應(yīng)系統(tǒng)（quorum sensing system，QSS）為銅綠假單胞菌（Pseudomonasaeruginosa，PA）毒力因子表達(dá)的重要調(diào)控系統(tǒng)。PA群體感應(yīng)（quorum sensing，QS）的引發(fā)與胞質(zhì)內(nèi)游離Ca2+濃度（cytosolic free Ca2+concentration，[Ca2+]i）的升高有關(guān)。鈣拮抗劑苯磺酸氨氯地平（amlodipine besylate，AML）可拮抗胞外的Ca2+內(nèi)流，調(diào)控PA的[Ca2+]i，從而抑制PA的QS。本研究探討AML的QS抑制活性及其可能機(jī)制。 方法 1細(xì)菌培養(yǎng) 采用平板劃線法活化并分離單一菌落，制備斜面工作管。挑取單一菌落分別于LB、MH、PTSB液體培養(yǎng)基搖床有氧培養(yǎng)。 2分組與給藥 設(shè)立正常對(duì)照組、0.5%二甲亞砜（dimethyl sulfoxide，DMSO）溶劑對(duì)照組、鈣離子載體A23187組（1μg·mL-1）及AML組（64、32、16μg·mL-1）。 3測(cè)定指標(biāo)及方法 3.1平板菌落計(jì)數(shù)法測(cè)定PA菌液中的活菌數(shù)，并繪制PA對(duì)數(shù)期“活菌數(shù)-OD600” 標(biāo)準(zhǔn)曲線，光電比濁法測(cè)定PA菌液中的活菌數(shù)（λ=600nm） 3.2光電比濁法測(cè)定AML對(duì)PA增殖的影響并繪制生長曲線（λ=600nm） 3.3微量肉湯稀釋法測(cè)定藥物對(duì)PA的最低抑菌濃度（minimun inhibitoryconcentration，MIC） 3.4蛋白水解酶試驗(yàn)測(cè)定PA蛋白水解酶的表達(dá) 3.5彈性蛋白酶試驗(yàn)測(cè)定PA彈性蛋白酶的表達(dá) 3.6綠膿毒素試驗(yàn)測(cè)定PA綠膿毒素的表達(dá) 3.7以Fura-2/AM為Ca2+熒光探針，熒光顯微鏡下觀察探針在PA的負(fù)載情況，并 用雙波長熒光分光光度法測(cè)定PA的[Ca2+]i 結(jié)果 1PA對(duì)數(shù)期菌液的“活菌數(shù)-OD600”標(biāo)準(zhǔn)曲線為y=0.0797x+0.0094，R2=0.9975 2AML對(duì)PA的MIC為512μg·mL-1 3AML（256、128μg·mL-1）可抑制PA的生長（P0.05）；AML（64、32、16μg·mL-1）對(duì)PA的生長無影響，，選定AML（64、32、16μg·mL-1）作為實(shí)驗(yàn)濃度 4AML（64、32、16μg·mL-1）可明顯抑制PA綠膿毒素的表達(dá)（P0.01）；可抑制蛋白水解酶和彈性蛋白酶的表達(dá)（P0.05） 5Fura-2/AM可成功負(fù)載于PA胞內(nèi)，可用340nm/380nm熒光強(qiáng)度比值R（F340nm/F380nm）測(cè)定PA [Ca2+]i 6AML（64、32、16μg·mL-1）可降低PA [Ca2+]i（P0.05） 結(jié)論 1AML（64、32、16μg·mL-1）可抑制PA毒力因子的表達(dá) 2AML抑制PA毒力表達(dá)的作用可能與干擾PA的鈣信號(hào)轉(zhuǎn)導(dǎo)系統(tǒng)調(diào)控PA的QS系統(tǒng)有關(guān)
[Abstract]:Objective (quorum sensing system QSS is an important regulatory system for the expression of virulence factor of Pseudomonas aeruginosa PA. The initiation of quorum sensing QS is related to the increase of intracellular free Ca 2 2 concentration (cytosolic free Ca 2 concentration, [Ca 2] I). The calcium antagonist, amlodipine besylate, antagonized the extracellular Ca 2 influx and regulated the [Ca 2] I of PA, thus inhibiting the QSs of PA. The aim of this study was to investigate the QS inhibitory activity of AML and its possible mechanism. Methods 1 A single colony was isolated and activated by plate crossing method to prepare inclined working tube. A single colony was isolated and cultured in a shaking bed on LBX MHH PTSB liquid medium. 2 the normal control group (dimethyl sulfoxide DMSO) was divided into two groups: normal control group (control group) with 0.5% dimethyl sulfoxide (DMSO) solvent control group. Calcium ion carrier A23187 (1 渭 g mL ~ (-1) and AML (64 ~ 32N ~ (16 渭 g 路mL ~ (-1). 3 the index and method 3.1 plate colony count method were used to determine the number of live bacteria in PA liquid, and to draw the standard curve of "live bacteria number -OD600" in the logarithmic phase of PA. Photovoltaic turbidimetry for the determination of the number of living bacteria in PA solution (位 _ (600) nm). 3. Photovoltaic turbidimetry to determine the effect of optoelectronic turbidimetry on the proliferation of PA and to draw a growth curve (位 _ (600) nm) for the determination of the minimal inhibitory concentration (minimun) of drugs on PA by broth dilution method. Inhibitory concentration MIC) 3.4 protein Hydrolase Assay determination of PA protein Hydrolase expression 3.5 elastase Test determination of PA elastase expression 3.6 expression of PA Pseudomonas aeruginosa by Pseudomonas aeruginosa Test 3.7 using Fura-2 / AM as Ca 2 fluorescence probe, The loading of the probe on PA was observed under a fluorescence microscope. Determination of [Ca 2] I of PA by dual wavelength fluorescence Spectrophotometry 1The standard curve of "number of living bacteria -OD600" in logarithmic phase of PA was 0.0797x 0.0094-R2O0.99752 AML against PA was 512 渭 g mL ~ (-1) 3AML (256128 渭 g mL ~ (-1). The growth of PA was inhibited (P0.05). AML (64N 32N 16 渭 g mL -1) had no effect on the growth of PA. AML (64N 32N 16 渭 g mL -1) could inhibit the expression of PA pyotoxin (P0.01), inhibit the expression of proteolytic enzyme and elastase (P0.05) and successfully load PA cell with 5Fura-2AM. 340nm/380nm fluorescence intensity ratio R (F 340 nm / F 380 nm) was used to determine PA [Ca 2] I 6 AML (64 4N 32U 16 渭 g mL -1), which could reduce PA [Ca 2] I (P0.05). Conclusion 1AML (64Nm / F 32U 16 渭 g mL -1) can inhibit the expression of PA virulence factor 2AML may inhibit PA virulence expression and interfere with PA virulence. The calcium signaling system of PA regulates the QS system of PA
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R96

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 孫瑋潔;王媛;沈立新;段康民;;銅綠假單胞菌群體感應(yīng)抑制物篩選系統(tǒng)的構(gòu)建及其應(yīng)用[J];生物工程學(xué)報(bào);2009年08期

2 朱春蓮;張立輝;;基因表達(dá)的鈣離子調(diào)控機(jī)制研究[J];江漢大學(xué)學(xué)報(bào)(自然科學(xué)版);2008年04期

3 邵梅,王洪梅,劉志洪,沈萍,蔡汝秀;鈣熒光探針Fura-2/AM對(duì)大腸桿菌細(xì)胞的負(fù)載行為[J];微生物學(xué)報(bào);2005年05期

4 任曉慧;王勝蘭;文瑩;楊克遷;;細(xì)菌中鈣信號(hào)的作用[J];微生物學(xué)報(bào);2009年12期

5 黃曉梅;趙子文;魏樹全;趙祝香;葉慧芬;;鈣拮抗劑對(duì)泛耐藥銅綠假單胞菌及鮑氏不動(dòng)桿菌體外聯(lián)合藥敏的影響[J];中華醫(yī)院感染學(xué)雜志;2011年11期

6 王文敏;徐志豪;;銅綠假單胞菌生物被膜形成的調(diào)控及治療對(duì)策研究進(jìn)展[J];浙江大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2010年01期

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