【作者】 杜飞燕；

【导师】 段争；

【作者基本信息】 河北医科大学 ， 内科学， 2012， 硕士

【摘要】 目的：研究表明可吸入颗粒物的增加可使肺炎的住院率增加，这在老年人及儿童中尤为明显。肺炎克雷伯杆菌是条件致病菌，是老年性肺炎主要致病菌之一。本研究建立PM_2.5暴露大鼠模型后予以肺炎克雷伯杆菌感染，通过检测PM_2.5暴露前后大鼠临床评分、肺脏病理、BALF中细胞计数、细菌菌落计数及细菌感染率，探讨了PM_2.5暴露对大鼠肺炎克雷伯杆菌感染的易感性和细菌清除率的影响；通过电镜检测大鼠气管粘液纤毛结构改变及血清中TNF-a及IL-6的含量，探讨了PM_2.5暴露导致大鼠呼吸系统细菌易感性增加的机制，以期对PM_2.5暴露导致的肺部感染性疾病的诊治提供理论依据。方法：清洁级雄性SD大鼠86只，按随机数字表法分为4组：①空白对照组；②生理盐水+肺克感染组；③PM_2.5+生理盐水组；④PM_2.5+肺克感染组；肺克感染指以肺炎克雷白杆菌标准菌株悬液浓度为1.0×105cfu/ml,以1ml/kg气管内滴入。PM_2.5暴露指PM_2.5以30mg/kg气管内滴入。各组动物根据评分标准进行临床评分，并分别于1天、7天处死部分动物，取材后测定相应指标。10%水合氯醛腹腔注射麻醉，股动脉放血处死动物并收集血液离心，吸取上清，应用ELASA方法检测血清中TNF-α和IL-6的含量。取右肺中下叶，10%中性福尔马林溶液固定，常规石蜡包埋，切片，HE染色，观察肺组织的病理形态变化并进行评分，对整个肺左叶行支气管肺泡灌洗，取支气管肺泡灌洗液，部分送细菌培养进行细菌菌落计数，部分离心后细胞沉淀悬浮于200μl生理盐水中，应用光学显微镜进行细胞计数。并取上段气管用扫描电镜观察气管黏膜上皮的细胞构成、纤毛情况。结果：1临床评分：空白对照组、NS+肺克感染组、PM_2.5+NS组和PM_2.5+肺克感染组1天的临床表现评分中位数和四分位数间距分别为4（1）、5（2）、5（1）、7（2）;7d的临床表现评分中位数和四分位数间距分别为1（1）、5（1）、4（2）、8（4）。即1天PM_2.5+肺克感染组动物的临床评分高于空白对照组（P<0.01）；而PM_2.5+NS组和NS+肺克感染组与空白对照组比较临床评分变化无统计差异（P>0.05）；PM_2.5+肺克感染组与NS+肺克感染组和PM_2.5+NS组相比动物的临床评分变化无统计学差异（P>0.05）。7天NS+肺克感染组、PM_2.5+NS组和PM_2.5+肺克感染组动物的临床评分均高于空白对照组（P<0.01），且PM_2.5+肺克感染组动物的临床评分高于NS+肺克感染组和PM_2.5+NS组（P<0.01）。空白对照组在第7天的临床评分较第1天下降有统计学意义（P<0.01）；NS+肺克感染组、PM_2.5+NS组和PM_2.5+肺克感染组不同时间临床评分均无明显改善（P>0.05）。2死亡率的比较：空白对照组、NS+肺克感染组、PM_2.5+NS组和PM_2.5+肺克感染组动物的7天总死亡率分别为：0%、30%、20%、46.7%。NS+肺克感染组、PM_2.5+NS组和PM_2.5+肺克感染组动物的死亡率明显高于空白对照组有统计学差异（P<0.0083）,且第7天PM_2.5+肺克感染组动物的死亡率也明显高于NS+PM_2.5组和NS+肺克感染组，差异有统计学意义（P<0.0083）；PM_2.5+肺克感染组第7天动物死亡率明显高于第1天（P <0.05）,而空白对照组、NS+肺克感染组和PM_2.5+NS组不同时间上动物死亡率无明显差异（P>0.05）。3肺脏组织病理学评分：1天和7天时NS+肺克感染组、PM_2.5+NS组、PM_2.5+肺克感染组肺组织病理分级高于空白对照组，均有统计学差异（P<0.05），且PM_2.5+肺克感染组较NS+肺克感染组和PM_2.5+NS组病理评分明显升高有统计学意义（P<0.05）。所有组第7天时较第1天的病理分级变化均无统计学意义（P>0.05）。4电镜气道粘液纤毛的改变：PM_2.5+NS组染毒1d时可见大鼠的气管纤毛呈杂乱、粘连、成束分布，摆动不良的外观,可见到其下的微绒毛。染毒7d时大鼠气管纤毛的粘连和倒伏状态更加严重,纤毛变短不规则，稀疏部分脱落，部分视野甚至纤毛缺失，可见新生的纤毛柱状上皮细胞。空白对照组大鼠气管柱状上皮纤毛的结构完整排列规则。5BALF中细胞总数及分类：结果表明1天和7天时NS+PM_2.5组、PM_2.5+NS组和PM_2.5+肺克感染组与空白对照组相比BALF中白细胞总数及中性粒细胞计数及比例均明显增多（P<0.05），且PM_2.5+肺克感染组BALF中白细胞总数及中性粒细胞计数及比例明显高于PM_2.5+NS组和NS+肺克感染组（P<0.05）。而NS+肺克感染组、PM_2.5+NS组和PM_2.5+肺克感染组第7天BALF中细胞总数及中性粒细胞计数及比例明显高于第1天（P<0.05）。而空白对照组不同时间细胞总数及分类变化没有统计学差异（P>0.05）。6血清中IL-6和TNF-α含量的变化：1天和7天时NS+肺克感染组、PM_2.5+NS组、PM_2.5+肺克感染组血清中IL-6和TNF-α含量均明显高于空白对照组（P<0.05），且PM_2.5+肺克感染组IL-6和TNF-α含量较PM_2.5+NS组和NS+肺克感染组明显升高有统计学意义（P<0.05）。NS+肺克感染组和PM_2.5+肺克感染组第7天IL-6和TNF-α含量明显高于第1天（P<0.05）,而PM_2.5+NS组与空白对照组不同时间点IL–6、TNF-α含量无明显变化。7BALF中细菌的检出率：NS+肺克感染组和PM_2.5+肺克感染组第1天细菌培养的阳性率分别为50%、75%，两组差别没有统计学意义（P>0.05）；7天时细菌培养的阳性率分别为25%、87.5%，PM_2.5+肺克感染组明显高于NS+肺克感染组（P<0.05）。两组组内不同时间点比较时细菌培养的阳性率均没有显著差异（P>0.05）。8BALF中肺炎克雷伯杆菌菌落计数：NS+肺克感染组和PM_2.5+肺克感染组1天时细菌菌落计数（Iogl0cfu/ml）的中位数和四分位数间距分别为：1.89（2.48）、1.00（2.02）；7天时细菌菌落计数（Iogl0cfu/ml）3.97（3.31）、4.70（1.02）。1天和7天时PM_2.5+肺克感染组细菌的生长数均明显高于NS+肺克感染组（P<0.05）。NS+肺克感染组和PM_2.5+肺克感染组菌落计数不同时间点比较时均没有明显的差别（P>0.05）。结论：1PM_2.5暴露可以导致大鼠肺部的炎症反应及气管的粘液纤毛系统功能受损。2PM_2.5暴露增加大鼠对肺炎克雷白杆菌感染的易感性；损害大鼠肺对肺炎克雷白杆菌的清除；加重感染所致的炎症反应。3PM_2.5暴露使大鼠对肺炎克雷白杆菌的易感性增加的机制涉及支气管的粘液纤毛系统功能受损以及炎症因子的相互作用。更多还原

【Abstract】 Objective:Epidemiological studies reveal increased hospitalization forpneumonia when levels of particulate matter in the air are increased,especially in children and the olderly population. Klebsiella pneumoniae is anopportunistic Gram-negative bacillus, which is one of a primary pathogenamong the olderly population with pneumoniae. We developed a rat model inwhich the animals were exposed to PM_2.5,followed by infection withKlebsiella pneumoniae. We sought to determine the effects of PM_2.5exposureon susceptibility to infection and bacterial clearance by examining clinicalappearance score, bacterial loads in bronchoalveolar lavage fluid （BALF）,histopathology assessment of lung, cell enumeration in BALF. We discussedmechanism of the increased susceptibility to infection by examining thecellulosity of tracheal membrane epithelium cells and condition of cilia byscanning electron microscope. In order to PM_2.5 expose lead to pulmonaryinfectious diseases provide the theoretical principle.Methods:86male SD rats of clean grade was divided into4groupsAccording to random number table:①c ontrol group;②normal saline+Klebsiella infection group;③PM_2.5+normal saline group;④PM_2.5+Klebsiella infection group；Klebsiella infection that tracheal instillation ofstandard strain of Klebsiella pneumoniae（1.0×105cfu/ml,1ml/kg）,PM_2.5exposure that tracheal instillation of PM_2.5by30mg/kg.Sacrificed some rats ofeach group after1stday and7thday separately, sampled and determinedcorresponding indexes.Anesthetized with an intraperitoneal injection of chloral hydrate10%,bloodletting from femoral and collected the blood, centrifugal,collect theblood and centrifuge it, drew out the supernatant fluid, and detected thecontents of TNF-αand IL-6by ELASA. Sacrificed the animals by open-chest, taken the middle and inferior lobe of right lung, fixed by10%neutral formalin,conventional paraffin-embedded, sliced, HE staining, observed thepathomorphism changes of lung tissue and scored, induced bronchoalveolarlavage of left lung bronchial by tracheal intubation, took BALF.Precipitatedcell was suspended in200μl saline, WBC count. Took the upper trachea, andthen observed the cellulosity of tracheal membrane epithelium cells andcondition of cilia, by scanning electron microscope（SEM）.Results:1Clinical score: The medians and quartile intervals of clinical manifestationscores of the first day of blank control group, NS+Klebsiella infection group,PM_2.5+NS group and PM_2.5+Klebsiella pneumoniae infection group were4（1）、5（2）、5（1）、7（2） respectively; The medians and quartile intervals ofclinical manifestation scores of the seventh day of were1（1）、5（1）、4（2）、8（4）.namely the clinical manifestation scores of the first day of the animals ofPM_2.5+Klebsiella infection group were higher than that of blank controlgroup（P<0.01）; and there were no statistics difference in the comparison ofthe clinical score change of PM_2.5+NS group、NS+Klebsiella infection groupand blank control group（P>0.05）; there were no statistics difference in thecomparison of the clinical score change of PM_2.5+Klebsiella infection groupand NS+Klebsiella infection group、PM_2.5+NS group（P>0.05）. The clinicalmanifestation scores of the seventh day of the animals of NS+Klebsiellainfection group, PM_2.5+NS group and PM_2.5+Klebsiella infection group wereall higher than that of blank control group（P<0.01）, and the clinicalmanifestation scores of the animals of PM_2.5+Klebsiella infection group washigher than those of NS+Klebsiella pneumoniae infection group andPM_2.5+NS group（P<0.01）. The decrease of the clinical scores of the seventhday of blank control group had statistical significance compared with those ofthe first day（P<0.05）; And there were no significant improvement in theclinical scores of NS+Klebsiella infection group、PM_2.5+NS group andPM_2.5+Klebsiella pneumoniae infection group at different times（P>0.05）.2Comparison of mortality rates: The total mortality rates of the seventh day of blank control group, NS+Klebsiella infection group, PM_2.5+NS group, andPM_2.5+Klebsiella infection group were0%、30%、20%、46.7%respectively.The mortality rates of the animals of NS+Klebsiella infection group,PM_2.5+NS group and PM_2.5+Klebsiella infection group were obviously higherthan that of blank control group, with statistical difference（P<0.0083）. Andthe mortality rate of the animals of the seventh day of PM_2.5+Klebsiellainfection group was also obviously higher than those of NS+PM_2.5group andNS+Klebsiella infection group, and the difference had statistical significance（P<0.0083）; The mortality rates of the seventh day of PM_2.5+Klebsiellainfection group was obviously higher than that of the first day（P<0.05）, butThere were no significant differences of the mortality rates of the animals ofblank control group, NS+Klebsiella infection group and PM_2.5+NS group atdifferent times（P>0.05）.3Lung histopathologic score: The histopathological grades of the first day andthe seventh day of NS+Klebsiella infection group, PM_2.5+NS group, andPM_2.5+Klebsiella infection group were higher than that of blank controlgroup, all had statistical differences（P<0.05）. And the significant rise ofpathologic scores of PM_2.5+Klebsiella infection group compared toNS+Klebsiella infection group and PM_2.5+NS group had statisticalsignificance（P<0.05）. All of the changes of pathological grades of the seventhday of all groups compared to those of the first day had no statisticalsignificance（P>0.05）.4Changes in airway mucociliary of electron microscopy: Tracheal cilia of thePM_2.5+NS group showed the appearance of disorderly arrangement, adhesion,sparse, distributed in beam, ciliary beat bad, and visible to the microvilli etc.And the symptoms aggravated gradually with the exposure timelengthened.The cilia was short, irregular and some of ecclasis,even lack of thecilia in partial vision,and neonatal ciliated columnar epithelial cells could befound.Columnar epithelial cilia in trachea of the control group rats werestructured and arrangement rules.5The total cell count of BALF and cell classification: The results showed that the total cell count of BALF and the count and percentage of neutrophils ofthe first day and the seventh day of PM_2.5+NS group, PM_2.5+NS group andPM_2.5+Klebsiella infection group increased significantly compared with theblank control group（P<0.01）, and the total cell count of BALF and the countand percentage of neutrophils of PM_2.5+Klebsiella infection group wereobviously higher than those of NS+PM_2.5group and NS+Klebsiella infectiongroup.（P<0.01） And the total cell count of BALF and the count andpercentage of neutrophils of the seventh day of NS+Klebsiella infectiongroup、 PM_2.5+NS group and PM_2.5+Klebsiella infection group wereobviously higher than those of the first day（P<0.05）. And there were nostatistical differences in the changes of the numbers of total cell count and cellclassification of blank control group at different times（P>0.05）.6The changes of contents of IL-6and TNF-α in serum: The results showedthat concentrations of IL-6and TNF-α in serum of the first day and theseventh day of NS+PM_2.5group, PM_2.5+NS group and PM_2.5+Klebsiellainfection group increased significantly compared with the blank controlgroup（P<0.01）, and concentrations of IL-6and TNF-α in serum ofPM_2.5+Klebsiella infection group were obviously higher than those ofNS+PM_2.5group and NS+Klebsiella infection group.（P<0.01）.Andconcentrations of IL-6and TNF-α in serum of the seventh day ofNS+Klebsiella infection group and PM_2.5+Klebsiella infection group wereobviously higher than those of the first day（P<0.05）. And there were nostatistical differences in the changes of concentrations of IL-6and TNF-α inserum of PM_2.5+NS group and blank control group at different times（P>0.05）.7The germ tested rate of BALF: The positive rates of the germiculture of thefirst day in the NS+Klebsiella infection group and PM_2.5+Klebsiella infectiongroup were50%,75%respectively, differences of these two groups had nostatistical significance（P>0.05）; The positive rates of the germiculture of theseventh day were25%、87.5%respectively,Those of the seventh day ofPM_2.5+Klebsiella infection group were obviously higher than those ofNS+Klebsiella infection group（P<0.05）. There were no significant differences in the positive rates of the germiculture of these two groups at different timepoints in intragroup comparisons（P>0.05）.8The colony count of Klebsiella pneumoniae of BALF: The medians andquartile intervals of the bacterial colony count（Iogl0cfu/ml） of the first day ofNS+Klebsiella infection group and PM_2.5+Klebsiella infection group were1.89（2.48）,1.00（2.02） respectively; and those colony counts of germicultureof the seventh day were3.97（3.31）,4.70（1.02） respectively. Those colonycounts of germiculture of the seventh day and the the first day ofPM_2.5+Klebsiella infection group were obviously higher than those ofNS+Klebsiella infection group（P<0.05）. And there were no significantdifferences of colony counts of NS+Klebsiella infection group andPM_2.5+Klebsiella infection group compared at different time points（P>0.05）.Conclusions:1The inflammatory response of the lungs and function impairment of thebronchus mucociliary system in the rats caused by exposure to PM_2.5.2The exposure to PM_2.5increased the susceptibility of the rats to Klebsiellapneumoniae infection; damaged the clearance of Klebsiella pneumoniae byrats’ lung; aggravated the inflammatory response caused by the infection.3The mechanism of enhances the susceptibility of the rats to Klebsiellapneumoniae caused by exposure to PM_2.5involving in function impairment ofthe bronchus mucociliary system and interaction of inflammatory factors.更多还原