【作者】 胡琼洁；

【导师】 熊盛道；

【作者基本信息】 华中科技大学 ， 内科学， 2010， 博士

【摘要】 目的：β-防御素-2在宿主防御病原微生物发挥着着重要作用,其不仅具有直接溶菌作用,同时也具有免疫调节的功能。本研究旨在探讨高表达β-防御素-2是否能减轻大鼠慢性铜绿假单胞菌的肺部感染。方法：借助线性聚乙烯亚胺(polyethylenimine,PEI)为载体,质粒编码的β-防御素2通过气道滴入的方法来介导质粒DNA的体内传递,48小时后再通过气道滴入藻酸盐包被的5×107c.f.u的铜绿假单胞菌。在感染后三天和感染后七天,观测大体病理,显微病理,细菌学计数,炎症的浸润程度及致炎细胞因子(IL-1β和TNF-α)和化学增活素(KC和MIP-2)的表达水平。结果：rBD-2质粒组大鼠三天生存率明显增高,感染后3天和7天的肺部细菌负荷明显下降。同时该组老鼠具有明显减轻的病理学改变相对于无任何处理的感染组和空质粒组。此外,相比无任何处理的感染组和空质粒组,在感染早期(感染后三天),rBD-2质粒组,中性粒细胞的募集和致炎细胞因子(IL-1β和TNF-α)和化学增活素(KC和MIP-2)蛋白的表达水平升高,然而在感染后期(感染后七天)其表达水平下降。结论：高表达β-防御素-2增加感染动物的存活率,降低了细菌的负荷。同时,高表达β-防御素-2增加早期中性粒细胞募集到感染部位和增强铜绿假单胞菌诱导的早期免疫反应。我们的研究结果表明,高表达β-防御素-2可能是慢性铜绿假单胞菌肺部感染的有效干预。目的：铜绿假单胞菌(PA)利用群体感应(QS)系统,一种细菌相互交流的信号系统,参与其多种致病因素.其中信号分子3OC12-HSL在PA的QS系统中起着重要作用。对氧磷酶1 (PON1)是一种内酯酶,可降解3OC12-HSL。本研究旨在观测高表达PON1是否对大鼠慢性铜绿假单胞菌的肺部感染有一定的保护作用及其可能的机制。方法：借助线性聚乙烯亚胺(PEI)为载体,通过气道滴注的方法介导PON1的高表达,48小时后再通过气道滴入藻酸盐包被的5×107c.f.u的野生型铜绿假单胞菌(PAO1),在感染后三天和感染后七天,分别观察大体病理,显微病理,细菌学计数,中性粒细胞的浸润程度(MPO).另再经气道滴入藻酸盐包被的QS报告基因的铜绿假单胞菌((PAO1-qsc102-lacZ),通过X-gal染色来反应β-半乳糖苷酶的量,从而间接反应了铜绿假单胞菌信号分子3OC12-HSL的量。结果：与无任何处理的感染组和空质粒组相比,PON1质粒组大鼠三天生存率无明显改变,7天的肺部细菌负荷和病理学改变明显下降。此外,相比无任何处理的感染组和空质粒组,在感染早期(感染后三天),PON1质粒组,中性粒细胞的募集明显升高,然而在感染后期(感染后七天)其下降。切片X-gal染色发现,PON1组未见明显的阳性的表达,而空质粒组X-gal染色见大量染成蓝色的阳性表达。结论：高表达PON1对大鼠慢性铜绿假单胞菌肺部感染有一定的保护作用,可能是通过作用于铜绿假单胞菌的QS系统,并增加早期中性粒细胞募集到感染部位来实现的。抑制QS可能作为治疗铜绿假单胞菌感染的新靶位,尤其是在对抗生素耐药的细菌感染病人。更多还原

【Abstract】 Objective:Beta-defensin-2 (BD-2) plays an important role in host defense against pathogenic microbe challenge by its direct antimicrobial activity and immunomodulatory functions. The present study aimed to determine whether genetic up-regulation of rat BD-2 (rBD-2) could ameliorate chronic Pseudomonas aeruginosa lung infection in rats.Methods:Plasmid-encoding rBD-2 was delivered to lungs in vivo using linear polyethylenimine at 48 h before challenging with seaweed alginate beads containing P. aeruginosa. Macroscopic and histopathological changes of the lungs, bacterial loads, inflammatory infiltration, and the levels of cytokines/chemokines [interleukin (IL)-1β, tumor necrosis factor (TNF)-α, kertinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2)] were measured at 3 and 7 days post-infection (p.i.).Results:The overexpression of rBD-2 resulted in a significant increase in animal survival rate (at 3 days p.i.), a significant decrease in bacterial loads in the lungs (at 3 and 7 days p.i.), and significantly milder lung pathology. In addition, the overexpression of rBD-2 led to increased infiltration of polymorphonuclear neutrophils (PMN), and elevated protein expression of cytokines/chemokines (IL-1β, TNF-α, KC and MIP-2) at the early stage of infection (at 3 days p.i.), at the same time as being dramatically decreased at the later stage of infection (at 7 days p.i.).Conclusions:Genetic up-regulation of rBD-2 increased animal survival rate, and reduced bacterial loads in lungs after bacterial infection. The overexpression of rBD-2 also modulated the production of several cytokines/chemokines and increased PMN recruitment at the early stage of infection. Our findings indicate that the enhancement of BD-2 may be an efficacious intervention for chronic P. aeruginosa lung infection. Objective:Pseudomonas aeruginosa uses quorum sensing, an interbacterial comm-unication system, to regulate gene expression. The signaling molecule N-3-oxo-dodecanoyl homoserine lactone (3OC12-HSL) is thought to play a central role in quorum sensing. Since 3OC12-HSL can be degraded by paraoxonase-1 (PON1), and PON1 expression protected against Pseudomonas aeruginosa lethality in Drosophila, suggesting that PON1 can interfere with quorum sensing in vivo, we hypothesized that the therapeutic potential of rat PON1 gene transfer in rats with P.aeruginosa-induced lung infection.Methods:Rats intratracheally infected with P.aeruginosa (PAO1) encapsulated in alginate beads were treated with a plasmid encoding PON1 or empty plasmid, which are complexed with linear polyethylenimine (L-PEI) and examined for survival of animals, lung bacterial load, inflammation, cytokine levels up to 3 and 7 days post-infection (p.i.). And rats intratracheally infected flies with a quorum-sensing reporter strain of P. aeruginosa (PAO1-qsc102-lacZ) that expressesβ-galactosidase under control of qsc102, which responds primarily to 3OC12-HSLResults:The overexpression of PON1 was associated with significant reductions in bacterial load and significantly milder lung pathology at 7 days post-infection. In addition, the overexpression of PON 1 led to increased infiltration of polymorphonuclear neutrophils (PMN) at the early stage of infection (at 3 days p.i.), at the same time as being dramatically decreased at the later stage of infection (at 7 days p.i.). And genetic up-regulation of PON 1 had less pronounced significant X-gal staining in rats.Conclusions:Overexpression of PON 1 ameliorates chronic P. aeruginosa lung infection in rats, probably through interfering with quorum sensing in vivo and increasing neutrophil recruitment an the early stage of P. aeruginosa infection. Novel therapeutic interventions aimed at directly regulating quorum sensing or PON activity may show promise for treatment with chronic P. aeruginosa lung infection.更多还原