环丙沙星胁迫蚯蚓的毒性效应及蛋白质组学研究

发布时间：2018-05-04 16:04

本文选题：环丙沙星 + 蚯蚓　；参考：《河北医科大学》2015年硕士论文

【摘要】：目的:氟喹诺酮类抗生素有良好的药物动力学性质,被广泛用于人类疾病治疗、畜牧和水产养殖活动,但其在人及动物体内的代谢率25%,所以大多数氟喹诺酮类抗生素以原形通过医疗废水、生活污水和人兽排泄物等途径进入环境。通过水环境迅速扩散至土壤后,被矿物质和有机物所吸附,并以盐酸盐的形式在土壤中形成累积,其生态毒理学的效应已成为我国乃至全球所必须面对的重大环境问题之一。本文拟通过对盐酸环丙沙星对蚯蚓急性和慢性毒性效应及蛋白表达谱的研究,探讨环丙沙星的暴露毒性和潜在暴露生物标志物,为早期诊断和评价环境中环丙沙星生态污染风险提供实验方法和数据参考。方法:参照经济合作与发展组织(OECD)制定的相关准则,蚯蚓的急性毒性试验采用滤纸接触染毒法(≤96 h)和人工土壤染毒法(≤14 d),慢性毒性试验采用人工土壤染毒法(14 d)。测定滤纸接触法中各个时间点(24 h,48 h,96 h)的半数致死量(LD50)和人工土壤法中各个时间点(12 h,1 d,2 d,7 d,14 d,28 d,56 d)的抗氧化酶(SOD,POD,CAT)活性。根据酶活性测定结果,选取关键的四个时间点(2 d,7 d,28 d,56 d)做苏木素-伊红(HE)染色病理组织切片,同时提取各组蚯蚓的总蛋白,利用双向电泳(2-DE)和基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)的技术与蛋白相关数据库的信息,寻找各个时间点的差异表达蛋白,然后选择部分蛋白,通过蛋白印迹法(Western blot),验证质谱鉴定结果。结果:在滤纸接触染毒试验中,暴露24 h、48 h和96 h后的LD50依次为2.20×10-1 mg·cm-2、1.60×10-1 mg·cm-2和1.12×10-1 mg·cm-2;在人工土壤染毒试验中,盐酸环丙沙星在最大试验浓度5 000 mg·kg-1时未引起蚯蚓死亡,说明人工土壤染毒法暴露7 d和14 d的LD50均大于5 000 mg·kg-1。酶活性测定结果为,1 000 mg?kg-1浓度组暴露2 d后的POD活性是对照组的2.48倍(P0.05),5 000 mg?kg-1组暴露56 d后的SOD活性是对照组的1.83倍(P0.05)。HE染色结果发现,2天和28天时肌纤维排列疏松,有一定的损伤,7天时肌纤维发生断裂,损伤比较严重,56天时,肌纤维排列紧密,没有明显的损伤。在蛋白质组学研究方面,每张胶图能被胶图分析软件PDQuset 8.0识别出大约1100个蛋白点,各组共发现110个蛋白点表达量有显著性(P0.05)变化(上调1.50倍或下调0.70倍),其中共36个蛋白点成功鉴定,共15种蛋白,在这36个点中,暴露于200 mg/kg盐酸环丙沙星2天后,有21个蛋白点发生上调,有6个蛋白点下调;7天后,有10个蛋白点发生上调,有7个蛋白点下调;28天后,有6个蛋白点发生上调,有12个蛋白点下调;56天后,有2个蛋白点发生上调,有5个蛋白点下调。这些点中,Actin蛋白占30.6%,Myosin蛋白占22.2%,在2天时多数有显著的上调,而在28天时多数有显著的下调,其次是一些与代谢途径相关的蛋白和一些推测的蛋白。各个时间点单独发生差异表达最大的蛋白分别是,Actin(2 d,3.52倍),Intermediate filament protein(7 d,3.55倍),Regucalcin(28 d,0.45倍)。应用Western blot技术对Myosin蛋白进行了验证。统计学分析结果表明,盐酸环丙沙星(200 mg/kg)胁迫赤子爱胜蚓2天时Myosin蛋白表达量相比对照组明显增高(P0.05)、7天时Myosin蛋白表达量相比对照组显著降低(P0.05)、28天和56天时Myosin蛋白表达量相比对照组没有变化(P0.05),这种变化趋势同蛋白质组学研究结果基本一致。结论:通过滤纸接触法胁迫蚯蚓,说明环丙沙星对蚯蚓存在一定致死毒性。在人工土壤暴露试验中,短期(≤14 d)暴露POD活性较为敏感,长期(14 d)暴露SOD活性较为敏感,两者的活性可作为有效的生物标志物对环丙沙星胁迫赤子爱胜蚓的毒性做出评价。通过200 mg/kg盐酸环丙沙星对赤子爱胜蚓进行2天、7天、28天和56天的胁迫,应用蛋白质组学方法,研究发现36个差异表达蛋白点,主要包含细胞骨架蛋白Actin蛋白和Myosin蛋白,这与HE染色结果和Western blot验证结果也基本一致。同时,还发现了与氧转运蛋白相关的胞外球蛋白。这些蛋白在一定程度上能够反映氧化应激损伤对于细胞骨架和肌肉收缩活动的破坏,可成为抗生素污染土壤潜在的分子暴露生物标志物。
[Abstract]:Objective: fluoroquinolones have good pharmacokinetic properties, and are widely used in human disease treatment, animal husbandry and aquaculture activities, but the metabolic rate in humans and animals is 25%, so most fluoroquinolones are in the original form through medical waste water, domestic sewage and human animal excreta and other ways to enter the environment. After the rapid diffusion of the environment to the soil, it is adsorbed by minerals and organic substances and is formed in the form of salt in the form of the soil. The effect of ecotoxicology has become one of the major environmental problems in China and even the world. This paper is intended to pass on the acute and chronic toxic effects and protein expression of ciprofloxacin hydrochloride on earthworms. Study on the exposure toxicity and potential exposure biomarkers of ciprofloxacin to provide experimental methods and data reference for the early diagnosis and evaluation of the risk of ciprofloxacin in the environment. Methods: the acute toxicity test of earthworms using filter paper contact dyeing method, based on the relevant guidelines formulated by the organization for economic cooperation and development (OECD). Less than 96 h) and artificial soil poisoning (less than 14 d), chronic toxicity tests were used by artificial soil (14 d). The determination of the antioxidant enzyme activity at each time point (24 h, 48 h, 96 h) in the filter paper contact method and the artificial soil method (12 h, 1 D, 2 D, 7 d, 14 d, 28 d, 28, 56) activity. The results were to select the key four time points (2 D, 7 d, 28 d, 56 d) to slice the pathological tissue of hematoxylin eosin (HE), and to extract the total protein of earthworms at the same time. The information of the technique of two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and protein related database were used to find each time point. Differentially expressed proteins, and then selected some proteins to verify the results of mass spectrometry identification by Western blot. Results: in the contact exposure test of filter paper, the LD50 after exposure to 24 h, 48 h and 96 h was 2.20 * 10-1 mg. Cm-2,1.60 * 10-1 mg cm-2 and 1.12 * 10-1 mg cm-2. In the artificial soil exposure test, ciprofloxacin hydrochloride was in the artificial soil test. The maximum experimental concentration of 5000 mg. Kg-1 did not cause earthworm death, indicating that the exposure of 7 d and 14 d by artificial soil exposure method was greater than 5000 mg kg-1. enzyme activity, and the POD activity after exposure to 2 D in 1000 mg kg-1 concentration group was 2.48 times as much as that of the control group (P0.05) and 1.83 times that of the control group after exposure 56. 05) the results of.HE staining showed that the muscle fibers were loosely arranged at 2 days and 28 days, and there were some injuries. At the time of 7 days, the muscle fibers were broken and the damage was more serious. At 56 days, the muscle fibers were closely arranged and there were no obvious damage. In the proteomics study, the glue map analysis software PDQuset 8 identified about 1100 protein points, each group could identify about 1100 protein points. A total of 110 protein points were found to have significant (P0.05) changes (up 1.50 times or 0.70 times down), of which 36 protein points were identified successfully, with a total of 15 proteins. At these 36 points, 2 days after 200 mg/kg were exposed to ciprofloxacin, 21 protein points were up-regulated and 6 protein points were down; 10 protein points were up-regulated after 7 days. There were 7 28 days later, 6 protein points were up and 12 protein points were down regulated. 56 days later, 2 protein points were up and 5 protein points were down. In these points, Actin protein accounted for 30.6%, Myosin protein accounted for 22.2%, and at 2 days the majority had significant up-regulation, and in 28 days, the majority had a significant downregulation, followed by some thanks to generations. Actin (2 D, 3.52 times), Intermediate filament protein (7 d, 3.55 times), Regucalcin (28 d, 0.45 times), respectively. The statistical analysis showed that ciprofloxacin hydrochloride (200) (200) was applied to ciprofloxacin (200). Mg/kg) the expression of Myosin protein was significantly higher in 2 days than that of the control group (P0.05), and the expression of Myosin protein was significantly lower than the control group at 7 days (P0.05). The expression of Myosin protein was not changed at 28 and 56 days (P0.05), and the trend was in accordance with the results of proteomics research. The contact method of filter paper stresses earthworms, indicating that ciprofloxacin has a certain lethal toxicity to earthworms. In the artificial soil exposure test, the exposure to POD activity is more sensitive in the short term (less than 14 d), and the exposure to SOD activity is more sensitive for a long time (14 d), and the activity of the two can be used as an effective biomarkers to evaluate the toxicity of ciprofloxacin. Valence. Using 200 mg/kg ciprofloxacin for 2 days, 7 days, 28 days and 56 days of the vermis of the red Eisenia vermis, the proteomics method was used to find 36 differentially expressed protein points, including cytoskeleton protein Actin protein and Myosin protein, which was also basically consistent with the results of HE staining and Western blot verification. Extracellular globulin related to oxygen transporter proteins, which can reflect the destruction of oxidative stress damage to cytoskeleton and muscle contraction to some extent, can be a potential biomarker for molecular exposure to antibiotics contaminated soil.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：X171.5

【共引文献】