本文選題：支氣管敗血波氏桿菌　切入點：PRDC　出處：《石河子大學》2017年碩士論文　論文類型：學位論文

【摘要】：在獸醫(yī)臨床上,支氣管敗血波氏桿菌(Bordetella bronchiseptica,Bb)造成豬的感染經(jīng)常發(fā)生,比如該病原菌是導致豬萎縮性鼻炎的主要因素。此外,Bb也是豬呼吸道綜合征(Porcine Respiratory Disease Complex,PRDC)的常見病原。然而,目前,Bb是如何突破豬呼吸系統(tǒng)的先天性免疫防御造成感染的,需要進一步研究。豬肺泡巨噬細胞(Porcine Alveolar Macrophages,PAMs),作為豬肺中主要的免疫細胞,起著豬肺“第一道防線”的作用。PAMs主要通過先天性的免疫受體比如Toll樣受體(TLRs)、C型凝集素受體(CLRs)或清道夫受體(SRs)識別病原微生物的侵入同時啟動先天性免疫反應。越來越多的研究顯示清道夫受體尤其是A類清道夫受體在病原菌的識別和吞噬中起著重要的作用。然而,目前對豬清道夫受體的研究還非常有限。進一步,PAMs是否可以通過A類清道夫受體如SRA和MARCO與Bb相互作用還不清楚?目的:為了進一步明確Bb在PRDC中的作用以及解析豬肺泡巨噬細胞與Bb的相互作用機制,本研究的目的主要有兩方面(1)明確Bb在PRDC的豬肺中的感染情況(2)解析PAMSs通過A類清道夫受體SRA和MARCO介導Bb的識別和吞噬。方法:(1)為了明確Bb在PRDC的豬肺中感染情況,首先,從上海不同的養(yǎng)豬場收集呼吸癥狀典型的病豬,剖檢后,利用TSA平板,對肺臟中的細菌進行分離和培養(yǎng)。隨后,對分離獲得的細菌,挑單菌落純化培養(yǎng)后,擴增16S rRNA基因序列并比對分析,對細菌進行鑒定;其次,對鑒定為Bb的細菌,利用PCR對相關(guān)毒力因子包括皮膚壞死毒素(DNT)、百日咳桿菌粘附素(PRN)以及鞭毛蛋白(fla B)進行分析;最后,對Bb分離株進行藥敏實驗,將純化好的Bb挑取單菌落液體培養(yǎng)至OD600約為0.6時,將菌液均勻涂布TSA,待菌液稍干時貼藥敏紙片,共選18種抗生素,18-24h后測量抑菌圈直徑并分析結(jié)果。(2)為了解析PAMs通過A類清道夫受體SRA和MARCO介導Bb的識別和吞噬,利用A類清道夫受體的阻滯劑分析A類清道夫受體與Bb的相互作用。首先:Bb分離株培養(yǎng)計數(shù)后,利用FITC染料對細菌進行標記,并使用顯微鏡檢測細菌標記的情況;利用流式細胞分析檢測不同處理情況下的PAMs對Bb的吞噬作用;其次,利用CHO-SRA、CHO-MARCO及CHO-vector細胞系,通過檢測熒光信號強度,進一步證實SRA和MARCO介導對Bb的識別和吞噬。結(jié)果:(1)呼吸道綜合征的豬肺中Bb的分離、鑒定及特性分析:首先,從15頭已鑒定為PRDC的豬肺中共獲得5株Bb分離菌株;其次,在對毒力因子分析中發(fā)現(xiàn)所有分離株的三種毒力因子檢測均為陽性。最后,對Bb分離株進行藥敏實驗,發(fā)現(xiàn)所有分離株對阿莫西林、頭孢噻呋、鏈霉素、氨芐西林等8種藥物高度耐藥,而對卡那霉素、美羅培南以及慶大霉素高度敏感,部分菌株對紅霉素、氟苯尼考、強力霉素以及阿米卡星耐藥。(2)PAMs通過A類清道夫受體SRA和MARCO介導Bb的識別和吞噬研究:首先,通過流式細胞分析,發(fā)現(xiàn)封閉PAMs上A類清道夫受體介導的吞噬,顯著地降低PAMs對Bb的吞噬,說明A類清道夫受體介導PAMs對Bb的識別和吞噬。進一步,我們的研究發(fā)現(xiàn)相對于CHO-vector,CHO-SRA或CHO-MARCO顯著地增強對Bb的吞噬。說明PAMs可通過A類清道夫受體主要是SRA和MARCO介導對Bb的識別和吞噬。結(jié)論:研究顯示Bb是引起PRDC的常見病原菌。同時,我們的研究顯示了PAMs可通過A類清道夫受體SRA和MARCO介導對Bb的識別和吞噬。該研究結(jié)果豐富了PAMs與細菌相互作用的機制,同時,為揭示PRDC的發(fā)病機制奠定基礎(chǔ)。
[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.

【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.61

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