【作者】 徐国鹏；

【导师】 熊盛道；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 第一部分乳铁蛋白对铜绿假单胞菌生长和毒力因子表达的调节及其机制目的研究乳铁蛋白对铜绿假单胞菌的杀菌性以及毒力因子表达和生物被膜形成的作用，同时探讨铁离子对其作用的影响。方法分别将乳铁蛋白溶液、FeCl₃溶液及乳铁蛋白与FeCl₃混合溶液分别加入到铜绿假单胞菌培养基(OD₆₀₀=0．05)中，以倍比稀释平板培养法检测活菌数评价其杀菌活性；以氯仿、盐酸萃取法定量检测绿脓菌素；以刚果红弹性蛋白检测弹性蛋白酶活性；结晶紫法定量检测生物被膜；以结晶紫染色法，在光镜下观察生物被膜形态。结果乳铁蛋白对铜绿假单胞菌具有杀菌活性，同时抑制绿脓菌素生成、弹性蛋白酶活性和生物被膜形成，其作用与浓度成正相关；FeCl₃促进铜绿假单胞菌生长，增加绿脓菌素生成和弹性蛋白酶活性，并进生物被膜形成；乳铁蛋白与铁结合后其杀菌性减弱，对绿脓菌素生成、弹性蛋白酶活性和生物被膜形成的抑制作用减弱。结论乳铁蛋白对铜绿假单胞菌具有杀菌活性，能抑制毒力因子的表达，其作用受铁离子的影响，可能部分地与其铁螯合能力有关。第二部分铁螯合剂对铜绿假单胞菌生长和毒力因子表达的调节及其机制目的研究铁螯合剂8-羟基喹啉、甲磺酸去铁胺对铜绿假单胞菌的杀菌性以及毒力因子表达和生物被膜形成的影响。方法分别将8-羟基喹啉溶液、甲磺酸去铁胺溶液及各自与FeCl₃混合溶液分别加入到铜绿假单胞菌培养基(OD₆₀₀=0．05)中，以倍比稀释平板培养法检测活茵数评价其杀菌活性；以氯仿、盐酸萃取法定量检测绿脓菌素；以刚果红弹性蛋白检测弹性蛋白酶活性；结晶紫法定量检测生物被膜；以结晶紫染色法，在光镜下观察生物被膜形态。RT-PCR法检测相关基因mRNA的表达。结果8-羟基喹啉、甲磺酸去铁胺均表现为抑制细菌生长、绿脓菌素合成、弹性蛋白酶活性和生物被膜形成，同时促进荧光素的表达；8-羟基喹啉螯合铁后对细菌生长、绿脓菌素合成、弹性蛋白酶活性和生物被膜形成的抑制作用增强，而甲磺酸去铁胺则反而促进细菌生长、绿脓菌素合成、弹性蛋白酶活性和生物被膜形成。结论铁螯合剂对铜绿假单胞菌的作用与其和铁的螯合能力和结构有关。铁饱和8-羟基喹啉可能通过新的机制对铜绿假单胞菌发挥作用。第三部分乳铁蛋白源性多肽和乳铁蛋白肽嵌合体对铜绿假单胞菌的作用目的研究乳铁蛋白源性多肽和乳铁蛋白肽嵌合体对铜绿假单胞菌的杀菌性以及毒力因子表达和生物被膜形成的影响。方法分别将LFcin溶液、LFampin溶液、LFchimera溶液及LFcin与LFampin混合溶液分别加入到铜绿假单胞菌培养基(OD₆₀₀=0．05)中，以倍比稀释平板培养法检测活菌数评价其杀菌活性；以氯仿、盐酸萃取法定量检测绿脓菌素；以刚果红弹性蛋白检测弹性蛋白酶活性；结晶紫法定量检测生物被膜；以结晶紫染色法，在光镜和荧光显微镜下观察生物被膜形态。结果LFcin、LFampin、LFchimera对铜绿假单胞菌具有杀菌活性，能抑制毒力因子的表达和生物被膜的形成，而且LFchimera的作用强于其组成多肽LFcin、LFampin及其混合物。结论乳铁蛋白源性多肽和乳铁蛋白肽嵌合体，尤其是LFchimera对铜绿假单胞菌具有良好的抗菌活性，有望用于治疗铜绿假单胞菌感染。更多还原

【Abstract】 PartⅠThe effect of lactoferrin and iron on the viability andexpression of virulence factors in Pseudomonas aeruginosaObjective To study the bactericidal activity of lactoferrin against Pseudomonasaeruginosa,and its effect on the expression of virulence factors and biofilm formation,atthe same time to explore the influence of iron on its activity.Methods Lactoferrin solution,FeC13 solution and lactoferrin plus FeC13 solution werediluted into a refolding solvent and added into the culture medium of Pseudomonasaeruginosa for evaluating the bactericidal activity respectively;pyocyanine quantificationassay was performed by extraction with chloroform and hydrochloric acid orderly;theelastase activity was detected by using Elastin Congo Red;biofilm quantification assay wasperformed by staining with crystal violet and spectrophotometer assay;biofilm morphologywas viewed by optical microscopy after being stained with crystal violet.Results Lactoferrin have shown the bactericidal activity against Pseudomonasaeruginosa,and inhibited pyocyanine production,elastase activity and biofilm formation,and these effects were positively correlated with their concentrations.FeC13 promoted thegrowth of Pseudomonas aeruginosa,and increased pyocyanine production and elastaseactivity and biofilm formation.These effects of lactoferrin were down-regulated byiron-binding.Conclusions Lactoferrin has bactericidal activity against Pseudomonas aeruginosa,caninhibit the expression of virulence factors.The effects of lactoferrin are correlated with itsiron-binding ability in part. PartⅡThe effect of iron chelators on the viability and expression ofvirulence factors in Pseudomonas aeruginosaObjective To study the bactericidal activity of 8-hydroxyquinoline(HQ)anddesferoxamine(DFO)against Pseudomonas aeruginosa,and their effects on the expressionof virulence factors and biofilm formation.Methods The solutions of 8-hydroxyquinoline,desferoxamine and one of them plusFeC13 were diluted into a refolding solvent and added into the culture medium ofPseudomonas aeruginosa for evaluating the bactericidal activity respectively;pyocyaninequantification assay was performed by extraction with chloroform and hydrochloric acidorderly;the elastase activity was detected by using Elastin Congo Red;biofilmquantification assay was performed by staining with crystal violet and spectrophotometerassay;biofilm morphology was viewed by optical microscopy after being stained withcrystal violet.Results HQ and DFO have shown the inhibition of bacterial growth,pyocyaninesynthesis,elastase activity and biofilm formation.However,iron-Saturated HQ has shownthe greater inhibition of bacterial growth,pyocyanine synthesis,elastase activity andbiofilm formation than apo-HQ,and iron-Saturated DFO has not shown the inhibition.Conclusions The effects of iron chelators on Pseudomonas aeruginosa are correlatedwith stability-constants for iron,and iron-Saturated HQ has greater inhibition of bacterialgrowth,pyocyanine synthesis,elastase activity and biofilm formation than apo-chelators. PartⅢThe effect of LF-derived peptides and LFchimera on theviability and expression of virulence factors in pseudomonasaeruginosaObjective To study the bactericidal activity of LF-derived peptides and LFchimeraagainst Pseudomonas aeruginosa,and their effects on the expression of virulence factorsand biofilm formation.Methods The solutions of LFcin,LFampin,LFchimera and LFcin plus LFampin werediluted into a refolding solvent and added into the culture medium of Pseudomonasaeruginosa for evaluating the bactericidal activity respectively;pyocyanine quantificationassay was performed by extraction with chloroform and hydrochloric acid orderly;theelastase activity was detected by using Elastin Congo Red;biofilm quantification assay wasperformed by staining with crystal violet and spectrophotometer assay;biofilm morphologywas viewed by optical microscopy after being stained with crystal violet.Results LFcin,LFampin,LFchimera have shown the bactericidal activity againstPseudomonas aeruginosa,and inhibited pyocyanine production,elastase activity andbiofilm formation,and these effects were positively correlated with their concentrations.The effects of LFchimera are greater significantly than LFcin,LFampin and their mixtures.Conclusions Lactoferrin-derived peptides and lactoferrin peptides chimera,especiallyLFchimera have the great antibacterial activity against Pseudomonas aeruginosa,arepromissory new compounds for a treatment for P..aeruginosa infections.更多还原