基于铜绿假单胞菌群体感应苯磺酸氨氯地平增加抗菌效应的机制研究

发布时间：2018-06-24 23:36

本文选题：铜绿假单胞菌 + 苯磺酸氨氯地平　；参考：《福建医科大学》2014年硕士论文

【摘要】：目的群体感应系统（quorum sensing system，QSS）为铜绿假单胞菌（Pseudomonasaeruginosa，PA）毒力因子表达的重要调控系统。PA群体感应（quorum sensing，QS）的引发与胞质内游离Ca2+浓度（cytosolic free Ca2+concentration，[Ca2+]i）的升高有关。钙拮抗剂苯磺酸氨氯地平（amlodipine besylate，AML）可拮抗胞外的Ca2+内流，调控PA的[Ca2+]i，从而抑制PA的QS。本研究探讨AML的QS抑制活性及其可能机制。方法 1细菌培养采用平板划线法活化并分离单一菌落，制备斜面工作管。挑取单一菌落分别于LB、MH、PTSB液体培养基摇床有氧培养。 2分组与给药设立正常对照组、0.5%二甲亚砜（dimethyl sulfoxide，DMSO）溶剂对照组、钙离子载体A23187组（1μg·mL-1）及AML组（64、32、16μg·mL-1）。 3测定指标及方法 3.1平板菌落计数法测定PA菌液中的活菌数，并绘制PA对数期“活菌数-OD600” 标准曲线，光电比浊法测定PA菌液中的活菌数（λ=600nm） 3.2光电比浊法测定AML对PA增殖的影响并绘制生长曲线（λ=600nm） 3.3微量肉汤稀释法测定药物对PA的最低抑菌浓度（minimun inhibitoryconcentration，MIC） 3.4蛋白水解酶试验测定PA蛋白水解酶的表达 3.5弹性蛋白酶试验测定PA弹性蛋白酶的表达 3.6绿脓毒素试验测定PA绿脓毒素的表达 3.7以Fura-2/AM为Ca2+荧光探针，荧光显微镜下观察探针在PA的负载情况，并用双波长荧光分光光度法测定PA的[Ca2+]i 结果 1PA对数期菌液的“活菌数-OD600”标准曲线为y=0.0797x+0.0094，R2=0.9975 2AML对PA的MIC为512μg·mL-1 3AML（256、128μg·mL-1）可抑制PA的生长（P0.05）；AML（64、32、16μg·mL-1）对PA的生长无影响，，选定AML（64、32、16μg·mL-1）作为实验浓度 4AML（64、32、16μg·mL-1）可明显抑制PA绿脓毒素的表达（P0.01）；可抑制蛋白水解酶和弹性蛋白酶的表达（P0.05） 5Fura-2/AM可成功负载于PA胞内，可用340nm/380nm荧光强度比值R（F340nm/F380nm）测定PA [Ca2+]i 6AML（64、32、16μg·mL-1）可降低PA [Ca2+]i（P0.05）结论 1AML（64、32、16μg·mL-1）可抑制PA毒力因子的表达 2AML抑制PA毒力表达的作用可能与干扰PA的钙信号转导系统调控PA的QS系统有关
[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
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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