Elafin重组气道上皮细胞对铜绿假单胞菌生物被膜的抗菌作用

目的探讨Elafin重组气道上皮细胞对铜绿假单胞菌(Pseudomonas aeruginosa,Pa)生物被膜(biofilm,BF)的抗菌作用。方法体外平板法制备Pa生物被膜模型,用银染法及扫描电镜鉴定。体外培养A549细胞,将pEGEP-N1-Elafin重组到A549细胞,倒置荧光显微镜观察转染情况。采用猪胰弹性蛋白酶(NE组)、铜绿假单胞菌(Pa组)上清、大肠埃希杆菌(E．coli组)上清分别诱导孵育重组细胞24 h,另设转染未含Elafin基因空载体的A549细胞作为对照组。将成熟BF载体放入4组中继续孵育8 h,扫描电镜观察4组BF结构的变化,并于第8小时用四甲基偶氮唑盐(MTT)法测定细菌存活数。结果扫描电镜下观察到3个诱导组的BF结构较对照组的BF明显松散,尤以Pa组和NE组为甚;NE组[(3．238±0．316)×106 CFU／cm2]、Pa组细菌数量[(3．317±0．247)×106 CFU／cm2]均较对照组[(5．946±0,453)×106 CFU／cm2]显著减少(P<0．01),E．coli组细菌数量[(5．138±0．391)×106 CFU／cm2]较Pa组和NE组减少,差异均有统计学意义(P<0．01)。3个诱导组BF细菌清除率与对照组相比分别为45．6％、44．2％和13．6％。结论Elafin重组气道上皮细胞能有效清除Pa生物被膜中的细菌,而Pa产物也是Elafin表达的有效诱导物。     

铜绿假单胞菌噬菌体的分离鉴定及其控制生物被膜的应用研究

目的 分离鉴定铜绿假单胞菌烈性噬菌体,研究噬菌体控制宿主菌形成牛物被膜的效率.方法 以铜绿假单胞菌临床菌株为指示菌,从不同环境样品中分离噬菌体;采用限制性内切酶图谱分析和宿主范围测定方法,对分离的噬菌体进行分类;利用透射电子显微镜对分离噬菌体进行形态学研究;以TJC729为指示菌,开展噬菌体控制牛物被膜形成的应用研究.结果 分别以14株铜绿假单胞菌临床分离菌株为指示菌,共分离得到13株烈性噬菌体,命名为C1～C13.利用限制性内切酶EcoR Ⅰ分析结果表明,13株噬菌体基因组均为双链DNA,并可被分成8组;宿主谱测定结果显示,c1和C13、C6和C7、C9和C11分别具有相同的宿主范围,其余7株噬菌体的宿主范围各不相同.随机挑选噬菌体C1进行形态学研究,发现噬菌体C1头部具有二十面体结构,尾部较长且无收缩性尾鞘,属于长尾噬菌体科.生物被膜控制实验结果显示,混合噬菌体能够较好地抑制TJC729生物被膜的形成.进一步实验结果显示,噬菌体C1、C10和C12分别与牛物被膜混合培养24 h后,牛物被膜的量分别下降到初始量的32.7%、57.6%、32.8%.结论 分离了13株铜绿假单胞菌烈性噬菌体,它们能够显著抑制宿主菌生物被膜的形成,并对生物被膜造成一定程度的破坏,为控制铜绿假单胞菌引起的感染提供了一个新方法.

Abstract：

Objective To isolate and classigy the bacteriophages specific to Pseudomonas aetuginosa and to investigate biofilm control efficaey of the isolated virulent phages.Methods With P. aeruginosa clinical strains as indicators.bacteriophages were isolated by screening difierent environmental samples.Classification of the isolated phages was done with the methods of restriction fragment analysis of phage genome and host range analysis.Transmission electron microscopy(TEM)was used in phage morphology study.In biogilm control tests,TJC729 was used as the jndicator strain to study the biofilm control efficacy of the isolated phages.Results Total 13 lytic phages specific to P.aeruginosa strains were isolated and named as C1-C13.According to the result of restriction fragment analysis.all 13 phages were double-stranded DNA viruses and could be divided into eight groups.Host range experiments were conducted with 5 laboratory strains and 12 clinical strains of P. aeruginosa.The same infection profiles were observed among phage C1 and C13,C6 and C7,and C9 and C11,respectively.While the remaining 7 phages each had different unique infection profile.Phage C1 was selected randomly to study its morphology.The obtained images showed that phage C1 had an icosahedral head with a non-contractile tail,belonging to the Siphoviridae family.Compared with the single phage,phage cocktail had the best effect on biofilm control.Further experiment results showed that phage C1.C10 and C12 can destroy biofilm after treatment of the biofilm for 24 h.The biofilm amounts were deceased to 32.7%,57.6%and 32.8%of the initial values,respectively.Conclusion Thirteen virulent phages specific to P. aeruginosa had been isolated.The phages could significantly inhibit the biofilm formation and had a certain degree of damage on the biofilm.The results suggested an alternative method for the treatments of P.aeruginosa infections.     

铜绿假单胞菌lasR rhlR基因缺陷对大鼠慢性肺部感染的影响

目的观察铜绿假单胞菌（Pseudomonas aeruginosa，Pa）PAO1野生型及群体感应（quorum sensing，QS）系统的lasR rhlR基因缺陷型Pα菌株人工生物被膜致病性的差异，及QS系统的lasR rhlR基因在Pα生物被膜感染致病过程中的地位。方法从大鼠支气管内直接予以Pα（菌种为PAO1野生型和PAO1 lasR rhlR基因缺陷型）海藻酸盐微菌粒悬液（10^9CFU／ml）攻击，建立PAO1野生型及QS系统的lasR rhlR基因缺陷型菌株人工生物被膜肺部感染动物模型。分别于感染1周和2周后评估各组大鼠的肺部病理学、细菌学及细胞因子（IL-4、IL-10）反应的变化。结果感染1周和2周后，PAO1野生型组的肺部病理学改变和细菌学改变均明显重于PAO1 lasR rhlR基因缺陷组（P〈0．001）；感染1周后PAO1野生型组肺部的IL-4、IL-10水平都高于PAO1 lasR rhlR基因缺陷组（P〈0．01，P〈0．05）；2周后PAO1野生型组肺部IL-10水平进一步升高（P〈0．01），明显高于PAO1 lasR rhlR基因缺陷组（P〈0．001）。结论PAO1野生型组在病程中引起持久和严重的肺部感染，激发了TH2样免疫反应，而PAO1 lasR rhlR基因缺陷组的肺部病变明显轻于PAO1组，说明QS系统的lasR rhlR基因在Pα肺部感染的建立及慢性化发展过程中发挥着重要作用。     

铜绿假单胞菌感染豚鼠后生物被膜形成的研究

目的 建立体内铜绿假单胞菌生物被膜模型，研究体内细菌生物被膜的组织学及细菌学特征。方法 通过吸入法使铜绿假单胞菌以气雾剂的形式吸入豚鼠肺内并生长定植，分别观察不同时期肺组织内细菌生物被膜的特征。结果 定植在肺内的铜绿假单胞菌以肉芽肿结节的形式存在，外周包绕类上皮细胞和成纤维细胞。结节内细菌被被膜基质所包绕并彼此连结，中间镶嵌宿主炎性细胞。接种后3周，肺内仍可见结节并培养出铜绿假单胞菌。结论 用吸入法可建立较稳定的铜绿假单胞菌肺感染生物被膜，其以肉芽肿结节的形式存在，宿主的反应细胞参与了生物被膜的形成。     

铜绿假单胞菌生物被膜形成过程中多糖合成相关基因表达的研究

目的 了解铜绿假单胞菌(Pseudomonas aerugirtosa,Pa)生物被膜形成过程中多糖生物合成相关基因在生物被膜形成中的表达,探讨其在生物被膜形成中的调控作用.方法 分别收集非黏液型铜绿假单胞菌PAO1的浮游菌及生物被膜菌,用实时荧光定量RT-PCR的方法对基因的表达进行相对定量分析.结果 多糖合成相关基因pslA、algD.pelA的mRNA在生物被膜菌中的相对表达量均高于浮游菌.结论 .pslA、algD,pelA的表达与铜绿假单胞菌生物被膜形成密切相关,在生物被膜形成中具有重要作用.     

铜绿假单胞菌生物被膜形成过程中多糖合成相关基因表达的研究

目的 了解铜绿假单胞菌(Pseudomonas aerugirtosa,Pa)生物被膜形成过程中多糖生物合成相关基因在生物被膜形成中的表达,探讨其在生物被膜形成中的调控作用.方法 分别收集非黏液型铜绿假单胞菌PAO1的浮游菌及生物被膜菌,用实时荧光定量RT-PCR的方法对基因的表达进行相对定量分析.结果 多糖合成相关基因pslA、algD.pelA的mRNA在生物被膜菌中的相对表达量均高于浮游菌.结论 .pslA、algD,pelA的表达与铜绿假单胞菌生物被膜形成密切相关,在生物被膜形成中具有重要作用.     

铜绿假单胞菌生物被膜形成过程中多糖合成相关基因表达的研究

目的 了解铜绿假单胞菌(Pseudomonas aerugirtosa,Pa)生物被膜形成过程中多糖生物合成相关基因在生物被膜形成中的表达,探讨其在生物被膜形成中的调控作用.方法 分别收集非黏液型铜绿假单胞菌PAO1的浮游菌及生物被膜菌,用实时荧光定量RT-PCR的方法对基因的表达进行相对定量分析.结果 多糖合成相关基因pslA、algD.pelA的mRNA在生物被膜菌中的相对表达量均高于浮游菌.结论 .pslA、algD,pelA的表达与铜绿假单胞菌生物被膜形成密切相关,在生物被膜形成中具有重要作用.     

铜绿假单胞菌生物被膜形成过程中多糖合成相关基因表达的研究

目的 了解铜绿假单胞菌(Pseudomonas aerugirtosa,Pa)生物被膜形成过程中多糖生物合成相关基因在生物被膜形成中的表达,探讨其在生物被膜形成中的调控作用.方法 分别收集非黏液型铜绿假单胞菌PAO1的浮游菌及生物被膜菌,用实时荧光定量RT-PCR的方法对基因的表达进行相对定量分析.结果 多糖合成相关基因pslA、algD.pelA的mRNA在生物被膜菌中的相对表达量均高于浮游菌.结论 .pslA、algD,pelA的表达与铜绿假单胞菌生物被膜形成密切相关,在生物被膜形成中具有重要作用.     

铜绿假单胞菌生物被膜形成过程中多糖合成相关基因表达的研究

目的 了解铜绿假单胞菌(Pseudomonas aerugirtosa,Pa)生物被膜形成过程中多糖生物合成相关基因在生物被膜形成中的表达,探讨其在生物被膜形成中的调控作用.方法 分别收集非黏液型铜绿假单胞菌PAO1的浮游菌及生物被膜菌,用实时荧光定量RT-PCR的方法对基因的表达进行相对定量分析.结果 多糖合成相关基因pslA、algD.pelA的mRNA在生物被膜菌中的相对表达量均高于浮游菌.结论 .pslA、algD,pelA的表达与铜绿假单胞菌生物被膜形成密切相关,在生物被膜形成中具有重要作用.     

铜绿假单胞菌生物被膜形成过程中多糖合成相关基因表达的研究

目的 了解铜绿假单胞菌(Pseudomonas aerugirtosa,Pa)生物被膜形成过程中多糖生物合成相关基因在生物被膜形成中的表达,探讨其在生物被膜形成中的调控作用.方法 分别收集非黏液型铜绿假单胞菌PAO1的浮游菌及生物被膜菌,用实时荧光定量RT-PCR的方法对基因的表达进行相对定量分析.结果 多糖合成相关基因pslA、algD.pelA的mRNA在生物被膜菌中的相对表达量均高于浮游菌.结论 .pslA、algD,pelA的表达与铜绿假单胞菌生物被膜形成密切相关,在生物被膜形成中具有重要作用.     

Circulating immune complexes in rats with autologous immune complex nephritis

Autologous immune complex nephritis (Heymann nephritis) was actively induced in rats by immunization with high (10 mg.) and low (1 mg.) doses of renal tubular epithelial antigen in complete Freund's adjuvant. The development of proteinuria and granular capillary wall deposition of IgG confirmed the previously well described membranous nephropathy which characterizes this experimental disease. Circulating immune complexes were demonstrated by both the fluid phase and solid phase C1q binding assays in both high and low dose experimental groups. The prevalence of such immune complexes was significantly greater in the experimental than in the control groups immunized with adjuvant alone or liver homogenate in adjuvant. The circulating immune complexes bound to C1q were 16 to 23 S in size and were proven to contain a renal tubular antigen. These data, in combination with the previous demonstration of renal tubular antigen and its antibody in kidneys from rats with autologous immune complex nephritis, are consistent with a circulating immune complex pathogenesis of this model of the actively induced autologous immune complex nephritis in rats.     

The relationship between hyperglycaemia and renal immune complex deposition in mice with inherited diabetes.

Kidney lesions were studied by light microscopy and immunofluorescence in diabetic (db/db) and obese (ob/ob) mutant mice. The db/db mutation was studied both on the C57Bl/KsJ genetic background (where it produces severe hyperglycaemia) and on the C57Bl/6J background (where hyperglycaemia is only mild). In all cases, more IgG, IgM and C3 were deposited in the renal glomeruli of mutant mice than in the glomeruli of normal (+/?) mice of equivalent age. First signs of immunoglobulin deposition occurred at a slightly younger age than first signs of C3 deposition or histological change (mainly mesangial thickening). Insulin deposits were occasionally seen in the glomeruli of older mutant mice and immunoglobulin eluted from diabetic mouse kidneys had anti-insulin activity. Increased anti-DNA activity was present in the serum of older mutants. In those mutants with severe hyperglycaemia, the macula densa and distal convoluted tubules also contained immunoglobulin deposits, probably derived from the glomerular mesangium. Urine from diabetic mice contained high molecular weight material reacting with antisera to Fab or kappa but not the Fc portion of immunoglobulin. We conclude that diabetic mice have immune complexes in the kidney containing antibodies against insulin and possibly other antigens. We find no evidence that hyperglycaemia itself is the direct cause of glomerular immune complex deposition, although there may be a link between hyperglycaemia and tubular dysfunction.     

细胞周期蛋白E在哮喘大鼠肺组织中的表达及其与气道重塑的关系

目的:通过测定哮喘大鼠肺组织中细胞周期蛋白E(cyclin E)的表达,研究cyclin E在哮喘气道重塑中的作用。方法:20只Wistar大鼠随机分为哮喘组和对照组,每组10只。用鸡卵清白蛋白(OVA)溶液致敏及激发复制哮喘模型。HE染色观察气道炎症;图像分析测量气道管壁厚度;流式细胞仪测定外周血单个核细胞细胞周期时相分布;免疫组化法检测cyclin E的表达情况;实时定量(real-time)RT-PCR测定肺组织中cyclin E mRNA水平表达;Western blotting法检测肺组织中cyclin E蛋白的表达。结果:哮喘组大鼠支气管管壁厚度较对照组增加(P0.01);哮喘组外周血单个核细胞S期、S+G2/M期百分率均较对照组增加,而G0/G1期百分率较对照组降低(均P0.01);哮喘组肺组织中cyclin E mRNA和蛋白水平的相对表达量较对照组高(均P0.01);肺组织中cyc-lin E蛋白的相对表达量与支气管管壁厚度呈正相关(P0.01)。结论:Cyclin E在哮喘大鼠肺组织中表达增加,其表达的上调可使细胞周期的G1期缩短,进而可能通过促进细胞的分裂增殖而参与哮喘气道重塑的发生发展。     

生物钟与肠道微环境、肠道免疫系统之间关系的研究进展

生物钟可以通过影响真核生物体内基因转录、改变生物氧化还原状态等方式保证机体每天进行正常的生长代谢节律波动,人体内的生物钟还能够调控肠道免疫细胞的增殖、分化及分泌功能.生物钟紊乱时会降低机体肠道免疫系统的抗病能力进而引起局部肠道黏膜损害或部分肠道炎症的发生.由大量微生物菌群组成的肠道微环境参与机体肠道黏膜保护、能量传递及营养代谢等生理过程,微环境发生改变时也会引起肠道病理性炎症反应.昼夜节律性改变也会改变正常的肠道微生态环境,使益生菌群数量下降并激活条件致病菌,产生大量有害代谢物质引起肠道炎症反应.生物钟、肠道菌群、肠道免疫系统之间存在着一定关系,但是它们之间的联系目前并不明确.明确生物钟、肠道微环境、肠道免疫防御、炎症性肠道疾病之间关系,从而探讨肠道疾病可能的发病机制,为炎症性肠道疾病治疗途径提供新的思路治疗途径提供新的思路.     

休克过程中小肠因素与肺损伤的关系研究

本实验观察休克时小肠因素在肺损伤中的作用。家兔随机分为SMAO休克组（n=11）和假休克组（n=8)，结果发现，肠系膜上动脉夹闭（SMAO）60分钟再灌注，肺毛细血管通透指数（PCPI）和肺组织丙二醛（MDA）含量增加，肺泡和支气管充血、水肿、出血及肺I型细胞结构损伤，肠病理损伤与肺损伤间有明显相关关系，但未见PaO2下降。用SMAO90分钟猫的SMV血血浆灌注离体大鼠肺标本（实验组，n=9;对照组，n=8)，致被灌注肺的肺动脉灌注压、PCPI和灌流液中MDA含量及乳酸脱氢酶活性显著升高，提示缺血小肠再灌注时释放的体液因素可直接引起肺损伤。     

Neuroendocrine and immune interactions with airway macrophages

Immune cells, including macrophages, are sources of many cytokines as well as a number of peptide hormones such as corticotropic releasing hormone and the endorphins. These mediators are involved in local immune regulation and may also function in an endocrine manner to influence the systemic hormonal response to stress. In turn, the major effectors of the systemic stress response, namely the glucocorticoids and the catecholamines, have been shown to modulate macrophage function. This review highlights some of what is known about neuroendocrine interactions with macrophages, and in particular, with airway macrophages, as modulation of airway macrophage function by neuroendocrine peptides is likely critical for the maintenance of lung homeostasis. The production of neuromediators (acetylcholine, catecholamines, sensory neuropeptides) and neuropeptide hormones (i.e. adrenocorticotropic hormone (ACTH) and beta-endorphin), from airway macrophages is also discussed. We have defined airway macrophages as those cells that reside in the airway compartment (i.e. versus the interstitium) and provide the first line of defence to inhaled particles.