清道夫受体A在小鼠巨噬细胞抗钩端螺旋体中的作用研究

发布时间：2018-04-11 18:13

本文选题：清道夫受体A + 钩端螺旋体　；参考：《上海交通大学》2015年博士论文

【摘要】：致病性钩端螺旋体所致的钩体病是一种世界范围传播的动物源性疾病。机体的固有免疫在清除钩体的过程中发挥了重要作用。但是,除了巨噬细胞的补体受体以外,尚未发现钩体的其它吞噬受体。清道夫受体A(SR A)等巨噬细胞表面的模式识别受体参与机体抗外来微生物的感染,结合前期SR A参与钩体吞噬的工作基础,我们提出巨噬细胞可通过SR A识别并吞噬钩体、参与固有免疫和调节炎症反应的假说。本研究发现,通过流式细胞术和激光共聚焦显微镜等方法,用特异性化学抑制剂Poly I和SR A单克隆抗体封闭巨噬细胞SR A,在缺乏血清补体调理的情况下,两者均能有效抑制小鼠巨噬细胞对钩体的吞噬。慢病毒转染SR A的HEK293T细胞与空载细胞相比,显著增强了对钩体的粘附作用。SR A基因敲除(SR A / )小鼠的巨噬细胞也表现出显著降低的吞噬能力。将抽提纯化的钩体LPS包被1μm小珠,发现WT巨噬细胞吞噬钩体LPS包被珠子的能力显著高于对对照珠子的吞噬,SR A / 小鼠的巨噬细胞对LPS包被珠子的吞噬则明显低于WT细胞。同样地,表达SR A的HEK293T细胞对LPS包被珠子的粘附显著高于空载细胞。同时,在钩体刺激下,SR A可与TLR4共定位,发生共受体作用,具有免疫调节效应。总的来说,这些结果表明在钩体感染过程中,巨噬细胞可通过SR A识别钩体LPS并吞噬钩体,能与TLR4发生共受体作用参与机体的固有免疫和调节炎症反应等过程。尿道致病性大肠埃希菌是尿路感染的首要致病菌。它的流行病学数据对临床治疗具有指导意义。本研究以2008年在上海收集的198株分离自尿路感染患者的大肠埃希菌为对象,通过药敏实验研究耐药表型,并基于PCR方法在分子水平检测耐药和毒力基因。采用脉冲场凝胶电泳(PFGE)和系统发育群分析菌株的同源性,并研究基因在各群体间的分布与相关性。药敏实验发现这些细菌具有极高的多重耐药(MDR)率(153/198),以及高达48.5%(96/198)的超广谱β内酰胺酶(ESBLs)阳性率,并且对青霉素、氟喹诺酮、磺胺类和第一、二代头孢菌素等高度耐药。分子水平的结果显示这些细菌以CTX-M-9群(70/96)耐药基因最为常见,其次为CTX-M-1群(27/96)。系统发育群D细菌占总数的42.4%(84/198),并在各系统发育群中拥有最高的ESBL率(50/84)和MDR率(75/84)。各毒力基因在系统发育群B2细菌中的比例显著偏高,但afa BC基因除外。对产ESBL的菌株进行PFGE分析,在67%的相似度下可被聚类为6个组。研究发现afa BC基因特定出现在系统发育群D和PFGE组I中。与其他报道不同,本实验发现afa BC与CTX-M-9群相关。总之,这些对细菌耐药与毒力因子的理解有助于临床开展针对尿路感染的经验治疗。
[Abstract]:Leptospirosis caused by pathogenic leptospirosis is a worldwide spread of animal disease.Innate immunity plays an important role in the clearance of leptospirosis.However, no other phagocytic receptors of Leptospira have been found except for the complement receptors of macrophages.The pattern recognition receptors on the surface of macrophages, such as scavenger receptor (A(SR), are involved in the prevention of infection by foreign microbes. In combination with the work basis of earlier stage of leptospira phagocytosis, we propose that macrophages can recognize and swallow leptospira by SR A.Hypotheses involved in innate immunity and regulation of inflammation.In this study, we found that Monoclonal antibodies (McAbs) of specific chemical inhibitors Poly I and SR A were used to block macrophage SR A by flow cytometry and laser confocal microscopy, without serum complement conditioning.Both of them could effectively inhibit the phagocytosis of Leptospira by mouse macrophages.HEK293T cells transfected with lentivirus significantly enhanced the adhesion to Leptospira. The macrophages of SR A knockout mice also showed significantly decreased phagocytosis.The purified Leptospira LPS was coated with 1 渭 m beads. It was found that the phagocytic ability of WT macrophages to LPS coated beads was significantly higher than that of WT cells to LPS coated beads.Similarly, the adhesion of HEK293T cells expressing SR A to LPS coated beads was significantly higher than that of unloaded cells.At the same time, under the stimulation of Leptospira, sarcoreceptor A could co-locate with TLR4, and had immunomodulatory effect.In general, these results suggest that macrophages can recognize LPS and phagocytosis of leptospirosis through SR A during the infection of Leptospira, and can co-receptor with TLR4 to participate in the process of innate immunity and regulation of inflammation.Urethral pathogenic Escherichia coli is the leading pathogen of urinary tract infection.Its epidemiological data have guiding significance for clinical treatment.In this study, 198 strains of Escherichia coli isolated from patients with urinary tract infection were collected in Shanghai in 2008. Drug resistance and virulence genes were detected at molecular level based on PCR method.The homology of the strains was analyzed by pulsed field gel electrophoresis (PFGE) and phylogenetic analysis, and the distribution and correlation of genes among different populations were studied.Drug sensitivity tests showed that these bacteria had a very high MDR rate of 153 / 198, and a high positive rate of Extended-spectrum 尾 -lactamases (ESBLs) up to 48.5 / 96 / 198), and were highly resistant to penicillin, fluoroquinolones, sulfanilamides and first and second generation cephalosporins.Results at the molecular level showed that the CTX-M-9 group 70 / 96)-resistant gene was the most common, followed by CTX-M-1 group 27 / 96.Phylogenetic group D accounted for 42.4% of the total, and had the highest ESBL rate of 50 / 84 and MDR rate of 75 / 84% among the phylogenetic groups.The proportion of virulence genes in B 2 bacteria was significantly higher, except for afa BC gene.The ESBL producing strains could be clustered into 6 groups under 67% similarity by PFGE analysis.It was found that afa BC gene was specific in group D and group I of PFGE.Different from other reports, we found that afa BC was associated with CTX-M-9 group.All in all, these understanding of bacterial resistance and virulence factors are helpful for clinical experience therapy for urinary tract infection.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R514.4

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