【作者】 张翠萍；

【导师】 马筱玲；

【作者基本信息】 安徽医科大学 ， 临床检验诊断学， 2012， 硕士

【摘要】 目的：金黄色葡萄球菌是引起人类各种感染性疾病的重要病原菌，携带P-V杀白细胞素(Panton-Valentine leukocidin, PVL)基因的金葡菌致病性增强，分布更广，可引起社区人群的严重感染。大量临床流行病学资料都强烈提示，PVL是金黄色葡萄球菌所致坏死性感染的重要毒力因素，但其致病机制尚不清楚。本课题通过体内试验观察重组PVL（rPVL）引起兔肺炎性损伤的变化，探讨多形核粒细胞(PMNs)、核转录因子-κB（NF-κB）和细胞因子在肺炎症性损伤中的作用，为阐释PVL的致病机制，寻求PVL相关肺炎症性损伤治疗方法提供新思路。方法：（1）动物模型的建立：取45只新西兰大白兔随机分为3组，分别为正常对照组、rPVL组和VCR+rPVL组（实验前用长春新碱预处理），每组15只。正常对照组使用PBS灌肺，其它两组均用rPVL灌肺。（2）BALF中PMNs计数、凋亡/坏死、ROS、LDH和LPI检测：造模后9h，采集支气管肺泡灌洗液（BALF），计数BALF中PMNs，流式细胞术（FCM）检测BALF中PMNs的凋亡/坏死率和活性氧（ROS）自由基释放量，比色法检测BALF上清中乳酸脱氢酶（LDH）含量，BCA法检测肺通透指数（LPI）。（3）肺组织湿干比及病理检测：造模后9h，收集肺组织检测湿重/干重比，并进行苏木精-伊红（HE）染色。（4）肺组织细胞因子和NF-κB检测：分别于造模后3h、6h和9h收集肺组织，每个时间段5只，ELISA方法检测肺组织匀浆上清中IL-6、IL-8、TNF-α和IL-10含量变化，免疫组织化学和Western blotting方法检测肺组织NF-κB p65蛋白的表达。结果：（1）与正常对照组比较，rPVL组BALF中PMNs数量显著增高，为(3.01±0.02)×106/ml vs（0.57±0.01)×106/ml；BALF中PMNs以晚期凋亡为主，晚期凋亡率显著增高，为（63.56±3.53）%vs（0.95±0.33）%；PMNs释放ROS增多，平均荧光强度（MFI）为（1.56±0.39）vs（0.41±0.03）；LDH含量和LPI均显著增高。VCR+rPVL组BALF中各项炎症指标均无明显增高。（2）与正常对照组比较，rPVL组肺湿重/干重比显著增高，为（6.45±0.35）vs（3.51±0.17），肺组织病理检查显示有弥漫性炎性细胞浸润、出血、水肿等。VCR+rPVL组肺组织湿重/干重比无明显增高，病理检查未见明显炎性变化。（3）rPVL组肺组织匀浆上清中IL-6、IL-8、TNF-α含量逐渐升高，IL-10含量于9h开始升高。VCR+rPVL组各种细胞因子含量均无明显升高。免疫组织化学方法检测结果显示，rPVL组NF-κBp65被激活转入核内，染色以核明显，且呈时间依赖性。VCR+rPVL组胞核中极少见或没有阳性免疫反应物质。Western blotting方法检测结果表明，rPVL组肺组织细胞核内NF-κBp65蛋白表达明显增加，且具有时间依赖性。结论：（1）本课题通过体内实验证实rPVL可以不依赖于细菌的生存和繁殖，单独引起肺组织炎症性损伤。（2）rPVL引起肺炎症性损伤依赖PMNs的招募和聚集，继而坏死，释放细胞毒素颗粒内容物和通过呼吸爆发释放活性氧代谢产物，引起肺组织损伤。（3）rPVL感染后，肺组织表现有NF-κB持续活化，促炎因子IL-6、IL-8、TNF-α含量逐渐升高，后期伴有抑炎因子IL-10升高。推测rPVL可能通过激活NF-κB，诱导促炎因子产生，促发炎症级联反应，引起肺组织损伤。更多还原

【Abstract】 Objective: Staphylococcus aureus, an important human pathogen, can cause a series ofinfections. The strains producing Panton-Valentine leukocidin(PVL) which contributeto the virulence, are widespread and cause a series of severe community associatedinfections. Compelling epidemiological data point to PVL as an important virulencefactor in S.aureus necrotizing infections, but its pathogenic mechanisms are notcompletely known. Therefore, in the present study, from animal experiment studies, westudy the mechanism of recombinant PVL (rPVL)-induced lung inflammation andinjury. To explore the role of polymorphonuclear leukocytes（PMNs）, nucleartranscription factor-κB (NF-κB) protein and cytokines in Panton-Valentine leukcocidin（PVL）–induced lung inflammation and injury. All these studies may establish thebasis for the further studies of its pathogenic mechanism and shed light on the treatmentof PVL-associated lung injury.Methods:(1) Establishment of animal model.45New Zealand white rabbits weredivided randomly into three groups, fifteen rabbits in each group. They were the controlgroup, rPVL treatment group and VCR+rPVL treatment group (vinblastine-treated).Then control group was treated with PBS, the other two groups were directly treatedwith endotracheal instillation of rPVL.（2） Study on the counts of PMN,apoptosis/necrosis, release of reactive oxygen species(ROS), LDH activity and lungpermeability index(LPI) in BALF. After9h post-infection, bronchoalveolar lavagefluid (BALF) was collected for counting PMNs, PMNs apoptosis/necrosis and therelease of ROS were analysed by flow cytometry(FCM), LDH activity was measured by colorimetric method, and LPI was detected by BCA.（3）Study on W/D ratio andhistopathology. After9h post-infection, the lung was collected from the rabbits fordetermine W/D ratio and H&E-stained.（4）Study on nuclear transcription factor-κB(NF-κB) protein and cytokines in lung injury. The lung was collected at3,6, or9hpostinfection, each time period had five rabbits. ELISA was performed to evaluate thelevels of expression of IL-6, IL-8, TNF-α和IL-10. NF-κB p65protein of the lung tissuewas assessed by immunohistochemistry and Western blotting method.Results:(1) Compared with control group, the counts of PMNs in BALF in rPVLtreatment group were significantly higher, which was (3.01±0.02)×106/ml vs (0.57±0.01)×106/ml. PMNs were dominated by late apoptosis, the apoptosis rate of PMNswas significantly higher, which was (63.56±3.53)%vs (0.95±0.33)%. The release ofROS was significantly higher, MFI was（1.56±0.39） vs（0.41±0.03）. LDH activity andLPI were all significantly higher. All inflammatory indicators in VCR+rPVL treatmentgroup were no significantly higher.(2) Compared with control group, W/D ratio inrPVL treatment group was significantly higher, which was (6.45±0.35) vs (3.51±0.17),histopathology study showed diffuse infiltration of inflammatory cells, hemorrhage,edema and other manifestations of lung injury. W/D ratio in VCR+rPVL treatmentgroup was no significantly higher, no inflammatory changes in lung.(3) The levels ofIL-6, IL-8and TNF-α in lung tissue in rPVL treatment group were increased gradually,and the level of IL-10was increased at9h postinfection. No cytokine in VCR+rPVLtreatment group was increased. The results of immunocytochemistry method showedthat in rPVL treatment group, increased NF-κB activity with nuclear localization in atime-dependent manner was observed. Western blotting method showed that expressionof NF-κB in nucleus in rPVL treatment group was increased gradually.Conclusion:(1) From animal experiment studies, these results indicate that rPVL caninduce lung inflammation and injury, independent of bacterial survival and replicationin the lung.(2) rPVL-induced lung inflammation and injury could be the result of recruitment and subsequent lysis of PMNs, which could damage the lung by releasingthe contents of cytotoxic granules and reactive oxygen metabolites.(3) After rPVLinfection, the expression of NF-κB and pro-inflammatory IL-6, IL-8, TNF-α in lungtissue were increased gradually, and lately IL-10was increased. We concluded rPVLcan induced the high expression of inflammatory cytokines by activating NF-κB protein,leading to lung tissue injury.更多还原