亚治疗量氟喹诺酮类药物加剧钩端螺旋体感染的分子机制研究

发布时间：2018-05-20 09:25

本文选题：钩端螺旋体 + 氟喹诺酮　；参考：《黑龙江八一农垦大学》2017年博士论文

【摘要】：钩端螺旋体病是由革兰氏阴性菌钩端螺旋体(以下简称钩体)引起的一种全球性、急性的人兽共患传染病。感染动物通过尿液排出致病性病原体,人和其他哺乳动物通过伤口或者黏膜组织接触污染的土壤和水源而发生感染。钩体病的主要临床症状表现为黄疸、血红蛋白尿、流产,多个重要脏器衰竭,甚至死亡等,严重危害了人和动物的健康。已有研究表明:亚治疗量的氟喹诺酮类药物环丙沙星能通过调节病原菌(葡萄球菌和大肠杆菌)的毒力和宿主的先天免疫来加重感染。本课题组研究发现:亚治疗量的氟喹诺酮类药物诺氟沙星治疗钩体病金黄地鼠导致实验组提前发病和死亡;体外抑菌试验发现最低抑菌浓度(MIC)以下的诺氟沙星能使钩体的形态变长。亚治疗量的诺氟沙星是否也调节了钩体的毒力,致使患病动物感染加重呢?如果是,那么其他的氟喹诺酮类药物在亚治疗量下是否也会有类似的现象发生呢?其内在的调控机制又是怎样的呢?对这些关键问题的科学解答,将有助于进一步阐明钩体的致病机制。本研究通过培养黄疸出血型钩体56601株,选择金黄地鼠为实验动物,构建钩体急性感染模型,选择氟喹诺酮类药物的代表性药物诺氟沙星和环丙沙星以不同剂量进行治疗,观察并记录21d动物的存活率。应用qPCR检测金黄地鼠血液、肝脏、肾脏和肺脏组织中钩体载量及细胞因子TNF-α和IL-1β的m RNA表达情况,并通过H.E.染色进行病理组织学观察。评价亚治疗量的氟喹诺酮类药物对钩体病的治疗效果,并分析其潜在的作用机制。体外应用不同浓度的诺氟沙星处理钩体,用暗视野显微镜和激光共聚焦显微镜观察钩体形态的变化,应用RT-qPCR检测钩体毒力蛋白Kdp A、Kdp B、Lip L21、Lip L41、Lip L71、Lip L32、Fla A、FlaB、Lig A、LigB的m RNA表达情况。并将处理后的钩体攻入金黄地鼠体内,观察动物的存活率。综合评价亚治疗量氟喹诺酮类药物治疗钩体病所存在的风险,并探讨其内在机制。研究结果表明:应用102~106钩体56601株感染金黄地鼠能产生稳定的半数致死结果,而该钩体对金黄地鼠的绝对致死量为107。应用亚治疗量的诺氟沙星和环丙沙星治疗感染钩体的金黄地鼠导致其存活率显著降低。qPCR检测发现,亚治疗量的诺氟沙星治疗组体内的钩体载量比未治疗组明显增多。同时,亚治疗量的诺氟沙星明显加重了肝脏、肾脏和肺脏的病理变化,并延迟了TNF-α和IL-1β在血液、肝脏、肾脏和肺脏中的基因表达。诺氟沙星对钩体56601株的MIC为1~2μg/ml,当诺氟沙星的浓度为0.25μg/ml时,钩体的形态明显增长。应用ge Norm软件,筛选出钩体的内参基因为Lip L41和Lip L71,通过2-(?)方法分析钩体毒力蛋白的表达,结果0.125μg/ml和0.25μg/ml的诺氟沙星均明显地上调钩体毒力蛋白FlaB和LigB的基因表达量。综上所述,本研究证实了亚治疗量的氟喹诺酮类药物治疗钩体病时存在的风险,该风险是通过亚治疗量的氟喹诺酮类药物延迟机体的炎症反应和上调钩体毒力蛋白FlaB和LigB的表达来实现的。
[Abstract]:Leptospirosis is a global, acute zoonosis caused by the Gram-negative leptospirosis (hereinafter referred to as the Leptospira). Infected animals infect the pathogenic pathogens through urine, and other mammals and other mammals are infected with contaminated soil and water through wound or mucosal tissue. Leptospirosis owners Clinical symptoms are jaundice, hemoglobinuria, abortion, multiple organ failure, and even death, which seriously harm the health of humans and animals. Studies have shown that the fluoroquinolone, ciprofloxacin, can aggravate the virulence of the pathogen (Staphylococcus and Escherichia coli) and the innate immunity of the host. The study found that norfloxacin, a subtherapeutic dose of fluoroquinolone, in the treatment of leptospirosis, caused early onset and death in the experimental group of Leptospira, and in vitro bacteriostasis test found that norfloxacin under the minimum inhibitory concentration (MIC) could make the shape of the Leptospira longer. If so, will the other fluoroquinolones be similar in subtherapeutic doses? What is the internal regulation mechanism? The scientific answers to these key questions will help to further clarify the pathogenesis of the leptospira. This study is made through the cultivation of jaundice. 56601 strains of blood type Leptospira were selected as experimental animals, the acute infection model of Leptospira was constructed, and norfloxacin and ciprofloxacin, a representative drug of fluoroquinolones, were treated with different doses, and the survival rate of 21d animals was observed and recorded. QPCR was used to detect the leptospira in the blood, liver, kidney and lung tissues of golden hamster. The expression of M RNA of cytokine TNF- alpha and IL-1 beta, and histopathological observation by H.E. staining. The therapeutic effect of fluoroquinolone on leptospirosis was evaluated and its potential mechanism was analyzed. The Leptospira treated with different concentrations of norfloxacin was used in vitro and the dark field microscope and laser confocal microscopy were used. The changes of the leptospiral morphology were observed by microscope, and the expression of leptospiral virulence protein Kdp A, Kdp B, Lip L21, Lip L41, Lip L71 were detected by RT-qPCR. The survival rate of the animal was observed by the treated Leptospira, and the survival rate of the animals was observed. The results showed that 56601 strains of 102~106 Leptospira infected with golden hamsters could produce a stable half lethal result, while the Leptospira's absolute lethal dose of norfloxacin and ciprofloxacin in the golden hamster had a significant reduction in the survival rate of the golden hamster. The low.QPCR test found that the load of the Leptospira was significantly increased in the group of norfloxacin treated group than that in the untreated group. At the same time, norfloxacin significantly increased the pathological changes in the liver, kidney and lungs, and delayed the gene expression of TNF- A and IL-1 beta in the blood, liver, kidney and lungs. Norfloxacin was 56601. The MIC of the plant was 1~2 g/ml. When the concentration of norfloxacin was 0.25 g/ml, the morphology of the Leptospira was obviously increased. Using GE Norm software, the genes of the Leptospira were selected to be Lip L41 and Lip L71. The expression of the Leptospira virulence protein was analyzed by 2- (?) method. The results showed that the 0.125 micron g/ml and 0.25 micron norfloxacin were all obviously up regulation of the Leptospira virulence protein. In summary, the present study confirms the risk of subleptospirosis in the treatment of subtherapeutic fluoroquinolones. The risk is to delay the inflammatory response by subtherapeutic fluoroquinolones and to increase the expression of FlaB and LigB of the leptospiral virulence protein.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S855

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