【作者】 罗启慧；

【导师】 程安春；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2012， 博士

【摘要】 肺炎链球菌是社区获得性肺炎最重要的致病原之一,为了进一步研究肺炎链球菌的致病机理,本实验对疑似感染肺炎链球菌猕猴进行解剖,并进行了微生物分离鉴定。同时采用H.E染色法对自发感染肺炎链球菌的猕猴消化系统、呼吸系统和免疫系统进行了组织病理学观察。肠道菌群在宿主的营养、代谢和宿主免疫保护等方面都起着非常重要的作用,而很多疾病的发生、发展也与肠道菌群结构的变化有着密切的联系。本论文对感染肺炎链球菌的猕猴和健康猕猴,采用不同类型分子生态学的方法研究了猕猴肠道菌群的组成变化。在此基础上,本实验以自发性感染肺炎链球菌猕猴为实验对象,采用免疫组织化学方法检测各系统在感染前后IL-6、IL-2、sIgA、IFN-y以及CD3蛋白质的表达情况。得到了如下实验结果：1、微生物分离鉴定结果：从心、肝、脾、肺组织及血液中分离到草绿色溶血菌株；胆汁溶菌实验和optochin实验结果表明分离菌株为肺炎链球菌,且为optochin敏感菌株；小鼠毒力实验结果表明本分离株对小鼠致病力强,为肺炎链球菌强毒株；16S rRNA序列分析表明与肺炎链球菌同源性高达99%以上,从分子水平上证实本实验分离到肺炎链球菌。结果表明本实验中采用的猕猴为强毒肺炎链球菌菌株感染猕猴。2、病理组织学结果：发现淋巴结出现大量巨噬细胞及粒细胞；脾血窦间隙增大,淋巴细胞增多；肺脏和气管均发生明显出血,肺泡隔增厚,有大量炎性细胞浸润；肝组织有广泛性的出血,部分肝细胞发生变性坏死；胃肠道黏膜层的腺体细胞发生广泛性的变性、坏死,腺体萎缩、结构破坏,黏膜层淋巴细胞大量增加；食管无明显病变。研究结果表明肺炎链球菌感染组猕猴出现了较为典型的组织病理学变化。3、PCR-DGGE技术分析结果：本研究通过对比2种DNA提取方法,提取到健康猕猴和肺炎链球菌感染后猕猴肠道菌群总DNA,利用细菌16S rRNA V3区通用引物进行扩增,产物经DGGE电泳,利用Quantity one图象分析软件对DGGE图谱进行分析；采用UPGAMA进行聚类分析；并计算Shannon-wiener多样性指数,Pielou指数,Margalef指数和Berger-Parker指数,数据采用SPSS19.0进行分析来测定群落结构形态。结果表明：试剂盒法提取的DNA纯度和数量上优于传统方法,DGGE图谱分析表明健康组猕猴肠道菌群结构相对稳定,PCR-DGGE条带数量以盲肠和结肠最多,其次是直肠、回肠和空肠,十二指肠最少,而肺炎链球菌感染组猕猴的各段肠道条带数显著减少(P<0.01),不同动物和肠段间菌群组成差别很大；健康组猕猴肠道细菌多样性指数、均匀度和丰度均高于感染组,与感染猕猴临床和病理表现相一致,感染后肠道微生物多样性降低。4、荧光定量PCR检测结果：采用SYBR Green I荧光定量PCR技术(FQ-PCR),建立6个菌属包括双歧杆菌、乳杆菌、拟杆菌、大肠杆菌、肠球菌和芽孢杆菌的实时荧光定量PCR检测方法,对健康组猕猴和感染组猕猴肠道内该6个菌属的数量和组成变化进行检测,结果表明：被检的6个菌属均为猕猴肠道中优势菌群,且其中双歧杆菌和乳杆菌含量最高(拷贝数对数分别为7.692±0.905,7.529±0.979),各个菌属在盲肠、结肠和直肠中含量高于肠道前段。肺炎链球菌感染后猕猴肠道优势菌群发生了较大变化,其中乳杆菌、双歧杆菌和拟杆菌拷贝数均显著下降(P<0.05)；芽孢杆菌和肠球菌数量也有一定程度下降,而大肠杆菌拷贝数有所增加,但未达到显著性差异。表明肺炎链球菌感染后猕猴肠道菌群失衡,与感染诱发的肠道炎症反应之间易形成恶性循环,本研究结果也对肠道菌群结构与机体健康关系提供新的启示。5、免疫组化检测结果：(1)消化系统(食管、胃、空肠、盲肠以及肝组织)检测,结果显示IL-6、 IFN-γ以及CD3感染后水平升高,IL-2、sIgA则呈现下降趋势。各种因子的表达主要集中在黏膜层。IL-6和IFN-γ在感染组的表达面积显著高于健康组(p<0.01),其中在空肠、盲肠和胃组织的表达更明显。IL-2在感染组的表达面积显著低于健康组(p<0.01),但是光密度值显示感染组显著升高(p<0.01)。CD3和sIgA阳性细胞表达面积在两组之间的差异并不明显,sIgA仅在空肠有显著降低,而CD3也仅在空肠和肝脏有显著升高(p<0.05)；CD3阳性细胞光密度值在肝脏和空肠明显增加,其他组织差异不明显。(2)呼吸系统(气管、肺)结果,感染组猕猴IFN-γ蛋白的阳性细胞面积在肺、气管和血管都明显增高(p<0.05),sIgA在感染组各组织的阳性表达也呈升高趋势,在气管的差异显著,IL-2蛋白的阳性表达面积略有增高,IL-6蛋白表达面积与健康组相比无明显差异；在气管,阳性细胞光密度值显示,和健康组比较,感染组IFN-γ、IL-2蛋白有明显增加(p<0.05),在血管和肺也仅有IgA和IFN-γ因子呈显著增加(p<0.05)。感染组猕猴肺脏和气管CD3蛋白表达产物的光密度值略高于健康组,但差异不明显,而其蛋白表达产物总面积则小于健康组,均达到了显著或极显著差异(P<0.05或P<0.01)。(3)免疫系统结果(脾和淋巴结),感染组猕猴脾脏内IFN-γ、IL-2蛋白的阳性表达面积都较健康组显著升高,而淋巴结的表达面积在两组间并无显著差异,除CD3外,各细胞因子和抗体都较健康组增加。感染组淋巴结内5种因子蛋白的表达光密度值均高于健康组,其中IL-6蛋白与健康组相比差异极显著(P<0.01), IFN-γ及IL-2蛋白表达量与健康组相比差异显著(P<0.05)。以上结果可看出,各种细胞因子和抗体的变化反应了肺炎链球菌对黏膜免疫系统的影响,这可能与肺炎链球菌的致病力相关联。整体上,与健康组比较,感染组IL-2在呼吸和免疫系统的表达面积增加而消化系统减少；IL-6表达面积在呼吸系统降低,免疫和消化系统增加；IFN-y在消化系统表达面积增加,而其他系统表达变化不一致；CD3在呼吸和免疫系统的表达面积减少,而消化系统整体增加；IgA的表达面积在呼吸和免疫系统的表达面积增加而消化系统减少。各个细胞因子相互联系,形成细胞因子网络,通过调节体液免疫和细胞免疫,相互协作抵御肺炎链球菌的侵害。更多还原

【Abstract】 Streptococcus pneumoniae is the main pathogen of Community acquired pneumonia. To investigate its pathogenesis, in this experiment we used rhesus monkeys as experiment animals to isolate and identify streptococcus pneumoniae. After obtaining the tissues, we detected histopathological changes in digestive system, respiratory apparatus and immune system by H.E staining. The intestinal flora plays a very important role in nutrition, metabolism and the host immune protection, and the structure of intestinal flora has very close relation with the development and changes in many diseases. In this assay, we chose two molecular ecology methods to detect structure of gut flora in healthy and infected with streptococcus pneumoniae rhesus monkeys. Expression changes of IL-6, IL-2, sIgA, IFN-y and CD3proteins were detected after rhesus monkey infected streptococcus pneumonia spontaneously by immunohistochemistry. The results in this study were as follows.1. Olive green hemolysis strains have been separated from blood and these organs including heart, liver, spleen and lung of infected rhesus monkeys. The results of bile solubility test and optochin-resistant test show that separated strain are streptococcus pneumoniae and resistant to optochin. The virulence test of mouse show the separated bacteria are virulent strains. Analysis of16S rRNA sequence indicates its homology is up to99%. All these tests show that those monkeys were infected with virulent streptococcus pneumoniae.2. Histopathological changes show that many macrophages and granulocytes are observed in lymph node; enlarged splenic sinusoid crevice and more lymphocytes; Lung and trachea with absolute bleeding, interalveolar septum thinckening and great inflammatory cell infiltrating; Hepatic tissue with catholic bleeding, a part of hepatocyte denaturation and mortification; Glandular cell in the stratum mucosum of gastrointestinal tract with catholic denaturization and mortification, accompanied by glandular organ atrophy and structure destroyed, lymphocytes of stratum mucosum increased greatly; Esophagus without obvious pathological changes. All the above changes indicate infected rhesus monkeys have typical histopathological changes caused by streptococcus pneumoniae.3. In this study, two methods were taken for DNA extract, and the result show extraction kit was better than normal methods in purity and production. Then16S rRNA V3region was amplified by common primers, and the PCR products were analyzed by DGGE electrophoresis. DGGE graph were analysed by Quantity one software(Bio-rad) and Cluster anlysis by UPGAMA. According to the data, we calculated Shannon-wiener diversity index, Pielou index, Margalef index and Berger-Parker index. These entire indexes were analysed by SPSS19.0for determinating structure morphology of bacteria community. The DGGE results indicate, healthy monkeys have stable enterobacteria, and the band number in cecum and colon are the most, next in rectum, ileum and jejunum, in duodenum the least. But in rhesus monkeys infected with streptococcus pneumoniae, the band number decrease significantly (P<0.01). Great difference in band number is observed among different monkeys and different intestines. Shannon-wiener diversity index, Pielou index, Margalef index and Berger-Parker index in guts of healthy monkey are higher than that of infected monkeys. This result consists with clinical and pathologic symptom which mean the diversity of gut flora decreased in infected rhesus monkeys,4. In this essay, SYBR Green Ⅰ FQ-PCR were established for detecting Bifidobacteria, Lactobacillus, Bacteroides, Escherichia coli, Enterococcus and Bacillus in alimentary tracts of rhesus monkeys. Results show the above six bacteria genus are the dominant flora in rhesus monkeys, the contents of Bifidobacteria and Lactobacillus among them are the most (logcopies were7.692±0.905and7.529±0.979, respectively). The contents of each genus in cecum, colon and rectum are higher than that in the former intestine. The dominant bacteria in infected monkeys change a lot. The logcopies of Lactobacillus, Bifidobacteria and Bacteroides decrease significantly (P<0.05), and logcopies of Bacillus and Enterococcus are lowered to some extent, but that of E.coli increase a little. These results revealed gut flora were disbalanced in rhesus monkeys infected with streptococcus pneumoniae, which is easy to form vicious cycle with inflammatory reaction provocated by infection. Also this research provides some new idea in the relation between gut flora and monkey’s health.5. Immunohistochemisty method was applied in this study for detecting protein expression, and all the results were as follows.(1) Results in Digestive system (including esophagus, stomach, liver, jejunum and cecum)(compared with normal group):The level of IL-6, IFN-y and CD3protein increased, IL-2and sIgA proteins decrease, proteins expression mainly localized in mucous layer. IL-6and IFN-y proteins positive cells areas are significantly higher than those in healthy monkeys (p<0.01), especially in jejunum, cecum and stomach. IL-2positive cells areas decrease significantly in infected group (p<0.01), but optical density value show that IL-2positive cells increase significantly in infected group (p<0.01). There are no obvious difference between infected and normal groups concerning CD3and sIgA proteins positive cells areas, only in jejunum, sIgA show decrease; in jejunum and liver, CD3positive cells increase significantly (p<0.05). Optical density value of positive cells express CD3proteins increase in liver and jejunum significantly (p<0.05).(2) Respiratory apparatus (trachea and lung) results:in the infected group, positive cells areas of IFN-y proteins increase significantly in lung, trachea and blood vessel (p<0.05); sIgA proteins also increase, but only obvious in trachea; IL-2proteins increase slightly, CD3and IL-6proteins show no significant difference. As for the optical density value of positive cells in infected group, IFN-y, IL-2proteins increase obviously in trachea (p<0.05), IgA anf IFN-y proteins increase obviously in blood vessel and lung (p<0.05), there are no significant difference in CD3and IL-6.(3) Immune system (spleen and lymph nodes) results. In infected group, positive cells areas of IFN-y, IL-2proteins increase significantly in spleen, and there are no significant difference in lymph nodes, except for CD3, the other cytokines all increase in infected group. All the five cytokines’ optical density value of positive cells areas increase in infected group, IL-6proteins show extreme significantly increase(p<0.01), IFN-y, IL-2proteins increase significantly (p<0.05).From above imunohistochemisty results, antibody expression point the influence of Streptococcus pneumoniae put on mucous immune system, which may involve in pathogenicity of Streptococcus pneumoniae. Compared with normal group, in infected group, the positive cells areas of IL-2proteins increased in respiratory apparatus and immune system and decrease in digestive system; IL-6proteins decrease in respiratory apparatus, and increase in Immune system and digestive system; IFN-y proteins increase in digestive system; CD3proteins decrease in Respiratory apparatus and Immune system and increase in digestive system; IgA proteins increased in respiratory apparatus and immune system and decrease in digestive system. All of the factors interact with each other and participate in regulating humoral immunity and cytoimmunity, and form a network to help host to resist disoperation resulting from Streptococcus pneumoniae.更多还原