支气管败血波氏杆菌分离鉴定及与猪肺泡巨噬细胞A类清道夫受体相互作用的研究

发布时间：2018-03-13 09:02

本文选题：支气管败血波氏杆菌　切入点：PRDC　出处：《石河子大学》2017年硕士论文　论文类型：学位论文

【摘要】：在兽医临床上,支气管败血波氏杆菌(Bordetella bronchiseptica,Bb)造成猪的感染经常发生,比如该病原菌是导致猪萎缩性鼻炎的主要因素。此外,Bb也是猪呼吸道综合征(Porcine Respiratory Disease Complex,PRDC)的常见病原。然而,目前,Bb是如何突破猪呼吸系统的先天性免疫防御造成感染的,需要进一步研究。猪肺泡巨噬细胞(Porcine Alveolar Macrophages,PAMs),作为猪肺中主要的免疫细胞,起着猪肺“第一道防线”的作用。PAMs主要通过先天性的免疫受体比如Toll样受体(TLRs)、C型凝集素受体(CLRs)或清道夫受体(SRs)识别病原微生物的侵入同时启动先天性免疫反应。越来越多的研究显示清道夫受体尤其是A类清道夫受体在病原菌的识别和吞噬中起着重要的作用。然而,目前对猪清道夫受体的研究还非常有限。进一步,PAMs是否可以通过A类清道夫受体如SRA和MARCO与Bb相互作用还不清楚?目的:为了进一步明确Bb在PRDC中的作用以及解析猪肺泡巨噬细胞与Bb的相互作用机制,本研究的目的主要有两方面(1)明确Bb在PRDC的猪肺中的感染情况(2)解析PAMSs通过A类清道夫受体SRA和MARCO介导Bb的识别和吞噬。方法:(1)为了明确Bb在PRDC的猪肺中感染情况,首先,从上海不同的养猪场收集呼吸症状典型的病猪,剖检后,利用TSA平板,对肺脏中的细菌进行分离和培养。随后,对分离获得的细菌,挑单菌落纯化培养后,扩增16S rRNA基因序列并比对分析,对细菌进行鉴定;其次,对鉴定为Bb的细菌,利用PCR对相关毒力因子包括皮肤坏死毒素(DNT)、百日咳杆菌粘附素(PRN)以及鞭毛蛋白(fla B)进行分析;最后,对Bb分离株进行药敏实验,将纯化好的Bb挑取单菌落液体培养至OD600约为0.6时,将菌液均匀涂布TSA,待菌液稍干时贴药敏纸片,共选18种抗生素,18-24h后测量抑菌圈直径并分析结果。(2)为了解析PAMs通过A类清道夫受体SRA和MARCO介导Bb的识别和吞噬,利用A类清道夫受体的阻滞剂分析A类清道夫受体与Bb的相互作用。首先:Bb分离株培养计数后,利用FITC染料对细菌进行标记,并使用显微镜检测细菌标记的情况;利用流式细胞分析检测不同处理情况下的PAMs对Bb的吞噬作用;其次,利用CHO-SRA、CHO-MARCO及CHO-vector细胞系,通过检测荧光信号强度,进一步证实SRA和MARCO介导对Bb的识别和吞噬。结果:(1)呼吸道综合征的猪肺中Bb的分离、鉴定及特性分析:首先,从15头已鉴定为PRDC的猪肺中共获得5株Bb分离菌株;其次,在对毒力因子分析中发现所有分离株的三种毒力因子检测均为阳性。最后,对Bb分离株进行药敏实验,发现所有分离株对阿莫西林、头孢噻呋、链霉素、氨苄西林等8种药物高度耐药,而对卡那霉素、美罗培南以及庆大霉素高度敏感,部分菌株对红霉素、氟苯尼考、强力霉素以及阿米卡星耐药。(2)PAMs通过A类清道夫受体SRA和MARCO介导Bb的识别和吞噬研究:首先,通过流式细胞分析,发现封闭PAMs上A类清道夫受体介导的吞噬,显著地降低PAMs对Bb的吞噬,说明A类清道夫受体介导PAMs对Bb的识别和吞噬。进一步,我们的研究发现相对于CHO-vector,CHO-SRA或CHO-MARCO显著地增强对Bb的吞噬。说明PAMs可通过A类清道夫受体主要是SRA和MARCO介导对Bb的识别和吞噬。结论:研究显示Bb是引起PRDC的常见病原菌。同时,我们的研究显示了PAMs可通过A类清道夫受体SRA和MARCO介导对Bb的识别和吞噬。该研究结果丰富了PAMs与细菌相互作用的机制,同时,为揭示PRDC的发病机制奠定基础。
[Abstract]:In the veterinary clinic, b.bronchiseptica (Bordetella bronchiseptica, Bb) caused by infected pigs often occur, such as the pathogen is a major cause of swine atrophic rhinitis. In addition, Bb is also the porcine respiratory syndrome (Porcine Respiratory Disease Complex, PRDC) of the common pathogens. However, at present, how is Bb break the swine respiratory system innate immune defense caused by infection, need further study. Porcine alveolar macrophages (Porcine Alveolar, Macrophages, PAMs), as the main immune cells in the lung, plays a role of.PAMs in pig lung "first line" mainly through the innate immune receptors such as Toll like receptor (TLRs), C C-type lectin receptors (CLRs) or scavenger receptor (SRs) and invasive identification of pathogenic microorganisms and activate innate immune responses. More and more studies show that scavenger receptor especially scavenger Receptor in the identification of pathogenic bacteria and phagocytosis plays an important role. However, the current research on pig scavenger receptor is also very limited. Further, whether PAMs can through the scavenger receptor such as SRA and MARCO and Bb interaction is not clear? Objective: to further clarify the role of Bb in PRDC and analysis of Porcine alveolar macrophages and Bb interaction mechanism, the main purpose of this study is two (1) Bb PRDC in clear lung infection (2) through the analysis of PAMSs scavenger receptor SRA and MARCO mediated Bb recognition and phagocytosis. Methods: (1) in order to clear, Bb in PRDC lung infection first, collect respiratory symptoms typical of infected pigs from different pig farms in Shanghai, necropsy after using TSA plate of bacteria in the lungs were isolated and cultured. Then the bacteria, pick a single colony of pure culture, amplification of 16S rRNA The gene sequence and comparative analysis were used to identify the bacteria; secondly, the identification of Bb bacteria, using PCR on the virulence factors including necrotoxin (DNT), pertactin (PRN) and flagellin (FLA B) were analyzed; finally, drug susceptibility test of Bb isolates, purification good Bb were single colony liquid culture to OD600 is about 0.6, the liquid is evenly coated TSA, when bacteria slightly dry paste drug, a total of 18 kinds of antibiotics, antibacterial circle diameter and the results of the analysis to measure 18-24h. (2) in order to identify the analytical PAMs by scavenger receptor SRA and MARCO guide Bb and phagocytosis, interaction analysis of scavenger receptor by Bb and scavenger receptor blockers. First: Bb isolates were cultured after counting marked bacteria using FITC dye, and the use of microscopy bacterial markers; using flow cytometry analysis. Phagocytosis test of different treatment under the condition of PAMs Bb; secondly, the use of CHO-SRA, CHO-MARCO and CHO-vector cell lines detected by fluorescence signal intensity, further confirmed that SRA and MARCO mediate the recognition of Bb and phagocytosis. Results: (1) from respiratory syndrome lung Bb, identification and analysis of characteristics: first of all, from the 15 head has been identified as PRDC lung were 5 strains of Bb isolated strains; secondly, in the analysis of virulence factors found in all three isolates detected virulence factors were positive. Finally, drug susceptibility test of Bb isolates, found all isolates of amoxicillin, ceftiofur, streptomycin ampicillin, 8 kinds of drugs are highly resistant to kanamycin, gentamicin meropenem and highly sensitive strains to erythromycin, florfenicol, doxycycline resistant and Amikacin. (2) PAMs by SRA and MARC scavenger receptor O mediated Bb recognition and phagocytosis of research: first, through flow cytometric analysis, find the closed scavenger receptor mediated phagocytosis of PAMs, PAMs significantly decreased the phagocytosis of Bb, indicating scavenger receptor mediated recognition of Bb PAMs and phagocytosis. Further, we study the relative to CHO-vector, CHO-SRA or CHO-MARCO significantly enhanced phagocytosis of Bb. PAMs by scavenger receptor SRA and MARCO is mainly mediated by the recognition of Bb and phagocytosis. Conclusion: the study shows that Bb is a common pathogen of PRDC. At the same time, our study showed that PAMs by scavenger receptor SRA and MARCO mediated recognition of Bb and phagocytosis. The research results enrich the mechanism, PAMs and bacteria interaction at the same time, to lay the foundation for revealing the pathogenesis of PRDC.

【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

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