本文選題：環(huán)丙沙星 + 蚯蚓　； 參考：《河北醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的:氟喹諾酮類抗生素有良好的藥物動(dòng)力學(xué)性質(zhì),被廣泛用于人類疾病治療、畜牧和水產(chǎn)養(yǎng)殖活動(dòng),但其在人及動(dòng)物體內(nèi)的代謝率25%,所以大多數(shù)氟喹諾酮類抗生素以原形通過(guò)醫(yī)療廢水、生活污水和人獸排泄物等途徑進(jìn)入環(huán)境。通過(guò)水環(huán)境迅速擴(kuò)散至土壤后,被礦物質(zhì)和有機(jī)物所吸附,并以鹽酸鹽的形式在土壤中形成累積,其生態(tài)毒理學(xué)的效應(yīng)已成為我國(guó)乃至全球所必須面對(duì)的重大環(huán)境問(wèn)題之一。本文擬通過(guò)對(duì)鹽酸環(huán)丙沙星對(duì)蚯蚓急性和慢性毒性效應(yīng)及蛋白表達(dá)譜的研究,探討環(huán)丙沙星的暴露毒性和潛在暴露生物標(biāo)志物,為早期診斷和評(píng)價(jià)環(huán)境中環(huán)丙沙星生態(tài)污染風(fēng)險(xiǎn)提供實(shí)驗(yàn)方法和數(shù)據(jù)參考。方法:參照經(jīng)濟(jì)合作與發(fā)展組織(OECD)制定的相關(guān)準(zhǔn)則,蚯蚓的急性毒性試驗(yàn)采用濾紙接觸染毒法(≤96 h)和人工土壤染毒法(≤14 d),慢性毒性試驗(yàn)采用人工土壤染毒法(14 d)。測(cè)定濾紙接觸法中各個(gè)時(shí)間點(diǎn)(24 h,48 h,96 h)的半數(shù)致死量(LD50)和人工土壤法中各個(gè)時(shí)間點(diǎn)(12 h,1 d,2 d,7 d,14 d,28 d,56 d)的抗氧化酶(SOD,POD,CAT)活性。根據(jù)酶活性測(cè)定結(jié)果,選取關(guān)鍵的四個(gè)時(shí)間點(diǎn)(2 d,7 d,28 d,56 d)做蘇木素-伊紅(HE)染色病理組織切片,同時(shí)提取各組蚯蚓的總蛋白,利用雙向電泳(2-DE)和基質(zhì)輔助激光解吸電離飛行時(shí)間質(zhì)譜(MALDI-TOF-MS)的技術(shù)與蛋白相關(guān)數(shù)據(jù)庫(kù)的信息,尋找各個(gè)時(shí)間點(diǎn)的差異表達(dá)蛋白,然后選擇部分蛋白,通過(guò)蛋白印跡法(Western blot),驗(yàn)證質(zhì)譜鑒定結(jié)果。結(jié)果:在濾紙接觸染毒試驗(yàn)中,暴露24 h、48 h和96 h后的LD50依次為2.20×10-1 mg·cm-2、1.60×10-1 mg·cm-2和1.12×10-1 mg·cm-2;在人工土壤染毒試驗(yàn)中,鹽酸環(huán)丙沙星在最大試驗(yàn)濃度5 000 mg·kg-1時(shí)未引起蚯蚓死亡,說(shuō)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。酶活性測(cè)定結(jié)果為,1 000 mg?kg-1濃度組暴露2 d后的POD活性是對(duì)照組的2.48倍(P0.05),5 000 mg?kg-1組暴露56 d后的SOD活性是對(duì)照組的1.83倍(P0.05)。HE染色結(jié)果發(fā)現(xiàn),2天和28天時(shí)肌纖維排列疏松,有一定的損傷,7天時(shí)肌纖維發(fā)生斷裂,損傷比較嚴(yán)重,56天時(shí),肌纖維排列緊密,沒(méi)有明顯的損傷。在蛋白質(zhì)組學(xué)研究方面,每張膠圖能被膠圖分析軟件PDQuset 8.0識(shí)別出大約1100個(gè)蛋白點(diǎn),各組共發(fā)現(xiàn)110個(gè)蛋白點(diǎn)表達(dá)量有顯著性(P0.05)變化(上調(diào)1.50倍或下調(diào)0.70倍),其中共36個(gè)蛋白點(diǎn)成功鑒定,共15種蛋白,在這36個(gè)點(diǎn)中,暴露于200 mg/kg鹽酸環(huán)丙沙星2天后,有21個(gè)蛋白點(diǎn)發(fā)生上調(diào),有6個(gè)蛋白點(diǎn)下調(diào);7天后,有10個(gè)蛋白點(diǎn)發(fā)生上調(diào),有7個(gè)蛋白點(diǎn)下調(diào);28天后,有6個(gè)蛋白點(diǎn)發(fā)生上調(diào),有12個(gè)蛋白點(diǎn)下調(diào);56天后,有2個(gè)蛋白點(diǎn)發(fā)生上調(diào),有5個(gè)蛋白點(diǎn)下調(diào)。這些點(diǎn)中,Actin蛋白占30.6%,Myosin蛋白占22.2%,在2天時(shí)多數(shù)有顯著的上調(diào),而在28天時(shí)多數(shù)有顯著的下調(diào),其次是一些與代謝途徑相關(guān)的蛋白和一些推測(cè)的蛋白。各個(gè)時(shí)間點(diǎn)單獨(dú)發(fā)生差異表達(dá)最大的蛋白分別是,Actin(2 d,3.52倍),Intermediate filament protein(7 d,3.55倍),Regucalcin(28 d,0.45倍)。應(yīng)用Western blot技術(shù)對(duì)Myosin蛋白進(jìn)行了驗(yàn)證。統(tǒng)計(jì)學(xué)分析結(jié)果表明,鹽酸環(huán)丙沙星(200 mg/kg)脅迫赤子愛(ài)勝蚓2天時(shí)Myosin蛋白表達(dá)量相比對(duì)照組明顯增高(P0.05)、7天時(shí)Myosin蛋白表達(dá)量相比對(duì)照組顯著降低(P0.05)、28天和56天時(shí)Myosin蛋白表達(dá)量相比對(duì)照組沒(méi)有變化(P0.05),這種變化趨勢(shì)同蛋白質(zhì)組學(xué)研究結(jié)果基本一致。結(jié)論:通過(guò)濾紙接觸法脅迫蚯蚓,說(shuō)明環(huán)丙沙星對(duì)蚯蚓存在一定致死毒性。在人工土壤暴露試驗(yàn)中,短期(≤14 d)暴露POD活性較為敏感,長(zhǎng)期(14 d)暴露SOD活性較為敏感,兩者的活性可作為有效的生物標(biāo)志物對(duì)環(huán)丙沙星脅迫赤子愛(ài)勝蚓的毒性做出評(píng)價(jià)。通過(guò)200 mg/kg鹽酸環(huán)丙沙星對(duì)赤子愛(ài)勝蚓進(jìn)行2天、7天、28天和56天的脅迫,應(yīng)用蛋白質(zhì)組學(xué)方法,研究發(fā)現(xiàn)36個(gè)差異表達(dá)蛋白點(diǎn),主要包含細(xì)胞骨架蛋白Actin蛋白和Myosin蛋白,這與HE染色結(jié)果和Western blot驗(yàn)證結(jié)果也基本一致。同時(shí),還發(fā)現(xiàn)了與氧轉(zhuǎn)運(yùn)蛋白相關(guān)的胞外球蛋白。這些蛋白在一定程度上能夠反映氧化應(yīng)激損傷對(duì)于細(xì)胞骨架和肌肉收縮活動(dòng)的破壞,可成為抗生素污染土壤潛在的分子暴露生物標(biāo)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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：X171.5

【共引文獻(xiàn)】

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