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研究生:呂庭薰
研究生(外文):LU, TING-HSUN
論文名稱:乙醯氨酚和布洛芬對 淡水長腳大蝦生理功能和抗致病菌感染力變化的影響
論文名稱(外文):Effect of Acetaminophen and Ibuprofen on Physiological Function and Resistance to Pathogen of Macrobrachium rosenbergii
指導教授:宋宏紅宋宏紅引用關係
指導教授(外文):SUNG, HUNG-HUNG
口試委員:楊喜男黃大駿
口試委員(外文):YANG, HSI-NANHUANG, DA-JI
口試日期:2017-05-25
學位類別:碩士
校院名稱:東吳大學
系所名稱:微生物學系
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:169
中文關鍵詞:乙醯氨酚布洛芬淡水長腳大蝦
外文關鍵詞:acetaminophenibuprofenMacrobrachium rosenbergii
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乙醯胺酚 (Acetaminophen, APAP) 及布洛芬 (Ibuprofen, IBU)為非類固醇類抗發炎藥物 (Non-steroid Anti-inflammatory Drugs, NSAIDs),也是目前台灣河川中偵測到的新興污染物質中最常見的人類用藥。對於這些藥物暴露於水體中,對水域生態甚至水產養殖可能造成的影響,目前瞭解甚少。本論文以台灣主要養殖之淡水蝦—淡水長腳大蝦 (Macrobrachium rosenbergii)俗稱泰國蝦,作為實驗動物,將其暴露於不同濃度之APAP與IBU不同天數後,針對免疫、抗氧化、壓力、解毒、肝損傷、以及呼吸生理等生理功能,測定10種基因轉錄的表現與6種酵素活性,並分析泰國蝦腸道菌相組成的變化及進行易感性實驗,以評估兩種藥物存在於水域環境對淡水蝦類的養殖可能造成的風險。
研究結果顯示,泰國蝦暴露於單一APAP下,高濃度(0.3 mg/L)APAP主要抑制基因轉錄的表現,包括:免疫相關原酚氧化酵素(prophenoloxidase)基因proPO以及解毒相關之穀胱甘肽硫基轉移酶(glutathione-S-transferase)基因GST;低濃度(0.03 mg/L)APAP抑制免疫相關基因proPO和α2-巨球蛋白(α2-Macroglobulin)基因α2-M轉錄的表現,而增加呼吸相關之細胞色素氧化酶亞基(Cytochrome oxidase subunit)基因cos轉錄表現量;單一IBU處理之泰國蝦,僅高濃度(0.2 mg/L)IBU顯著抑制免疫相關基因α2-M的轉錄表現;低濃度(0.02 mg/L)IBU則無顯著影響(p<0.05);兩種藥物同時存在影響免疫相關基因proPO以及抗氧化相關銅鋅依賴型超氧歧化酶(Superoxide dismutase- Copper, Zinc)基因SOD-Cu,Zn,轉錄表現上升,而免疫相關之細胞黏附因子(peroxinectin)基因pon和α2-M、以及呼吸相關之基因cos的轉錄表現量則降低。酵素活性的研究結果顯示,單一APAP下,能提升免疫相關之酚氧化酵素(phenoloxidase, PO)活性以及降低肝損傷相關之麩胺酸草酸轉胺酶(glutamic oxaloacetic transaminase, GOT)和丙酮酸酸草酸轉胺酶(glutamic pyruvic transaminase, GPT)活性;單一IBU下,促使免疫相關之PO和解毒相關之GST活性增加,且降低肝損傷相關之GOT和GPT活性;兩種藥物同時存在,能立即誘發血漿中的proPO被催化成PO,引起發炎反應,降低抗氧化相關之榖胱甘肽過氧化物酶(Selenium-dependent Glutathione peroxidase, GPx)活性,並使肝損傷相關之GOT和GPT活性增加。其他生理功能酵素活性則影響不一致。兩種藥物同時存在對泰國蝦部分的生理功能之影響效應與單一存在的效應有差異,包括對免疫反應的包外POS 活性具有協同作用,對呼吸基因cos轉錄表現及抗氧化酵素GPx活性產生拮抗性干擾,對肝損傷酵素GOT活性為增效性,GPT活性則為拮抗。此外,不論暴露於APAP和IBU單一或同時存在池水,泰國蝦經致病菌感染後的死亡率低於對照組,其腸道菌相的組成與對照組不同,且浸泡後第10天出現在腸道中的致病菌Lactococcus garviae在總菌相所佔之比例低於浸泡前。綜合本論文所有的結果推測,目前環境殘留的APAP及IBU對蝦子是安全的並對抗病力具正效應,對目前台灣淡水蝦類的養殖應不具威脅的。
Acetaminophen (APAP) and ibuprofen (IBU) which are non-steroid anti-inflammatory drugs (NASIDs), are the most common emerging pollutants of the NASIDs in rivers in Taiwan. The impacts of the residues of the both drugs on aquatic ecosystem and aquaculture are poorly understood. To assess the potential risk of APAP and IBU for cultured freshwater prawn, in this study, Macrobrachium rosenbergii, which a major cultured freshwater prawn in Taiwan, was used as the experimental animal and exposed to different concentrations of APAP and IBU for 10 days, respectively. After immersion, different physiological functions of the kinds of gene transcription levels and six kind of enzyme activities, including immunity, antioxidation, stress, detoxification, respiration, and hepatopancreas damage, were detected by semi-quantitative RT-PCR and enzymes assay kits, respectively. In addition, the change of bacterial flora in feces of prawn and the susceptibility of prawn to the infection of pathogens were examined.
The results from prawns immersed in APAP showed that, the transcriptional levels of two genes were inhibited at APAP with high concentration (0.3 mg/L), including the immunity-related gene prophenoloxidase (proPO) and one detoxification-related gene glutathione-S-transferase (GST); at low concentration (0.3 mg/L) of APAP, three kinds of gene transcription can be influenced, including reducing the mRNA amounts of both proPO and α2-Macroglobulin (α2-M) and one respiration-related gene cytochrome oxidase subunit (cos). After prawns immersed in IBU, only the gene α2-M was significantly inhibited at high concentration (0.2 mg/L)(p<0.05) and all target genes were not significantly changed at lower concentration (0.02 mg/L). The results from prawns immersed in the pond water containing mixing with an equal concentration of APAP and IBU at at 0.015 mg/L each showed that the mRNA amount of the gene proPO and the antioxidation-related gene superoxide dismutase-copper, zinc (SOD-Cu, Zn) were increased; however, those of the immunity-related gene peroxinectin (pon), and α2-M, and the respiration-related gene cos were reduced.
As for the effect of the both drugs on enzyme activity of different physiological functions, three kinds of activities of enzyme activities were influenced after prawn exposed to APAP, including increasing of phenoloxidase (PO) activity, and reducing activities of hepatopancreas damage-related glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT). When the prawns were exposed to IBU, Shrimps immersed alone IBU, the increase of PO activity and the detoxification related glutathione-S-transferase (GST) activity, and the decrease of the activity of GOT and GPT were detected. After prawns exposed in mix of APAP and IBU, the proPO converted into PO were immediately elicited in plasma, the activity of the antioxidation-related enzyme, selenium-dependent Glutathione peroxidase (GPx) was reduced, and the activities both GOT and GPT activity were increased. According the above results, this study found that, for some physiological function genes or enzymes, APAP and IBU affected each other, including the synergy for PO activity, the antagonism for the gene cos expression, the potentiation for GOT activity, and the antagonism for GPT activity. Furthermore, the results from pathogen-challenged prawns challenged with pathogen Lactococcus garviae were found that the mortality of all experimental groups was lower than that of control. Finally, the comparison the gut bacterial composition between control group and experimental group showed that the composition was various in different treated groups, but that of the same treated-groups was similar; as well as, after exposure for 10 days. The lower proportion of the pathogen L. garviae in the total gut bacteria from the all experimental groups than that of the control groups was detected. Based on all the results, this study speculates that, the experimental concentrations of APAP and IBU not only are safety to freshwater prawn but had a positive effect on resistance to infection.Therefore, the environmental concentration of APAP and IBU in Taiwan rivers should not threat to freshwater shrimp farming in Taiwan.

目錄
中文摘要
壹、前言
貳、文獻回顧
一、 非類固醇類抗發炎藥物(Nonsteroid Anti-inflammatory Drugs, NSAIDs )
(一) 非類固醇類抗發炎藥物的流佈及管理
(二) 乙醯氨酚(APAP)及布洛芬(IBU)對生物的影響
二、 台灣養殖蝦類的現況
(一) 淡水長腳大蝦養殖面臨的問題
三、 蝦類生理指標(physiological indicators)
(一) 免疫
(二) 抗氧化
(三) 壓力
(四) 解毒作用
(五) 肝胰臟損傷
(六) 呼吸
(七) 腸道菌相
參、實驗目的與策略
肆、材料和方法
一、 實驗用蝦條件及養殖環境
二、 非類固醇類抗發炎藥物之製備
三、 淡水長腳大蝦實驗模式及採樣時間
(一) 不同生理功能的基因表現與活性測定之採樣
(二) 易感性的藥物處理與感染的時間
(三) 腸道菌相分析之糞便採樣
四、 蝦血淋巴液的製備
五、 泰國蝦生理功能基因之表現
(一) 蝦血球總 RNA 的萃取
(二) 反轉錄實驗
(三) 基因引子之設計與實驗條件
(四) 半定量聚合酶鏈鎖反應測定
六、 泰國蝦生理活性測定
(一) 蝦血漿及血球細胞萃取液的製備與蛋白質濃度測定
(二) 免疫之酚氧化酵素(phenoloxidase, PO)
(三) 抗氧化之超氧化物歧化酶(superoxide dismutase, SOD)
(四) 抗氧化之穀胱甘肽過氧化物酶(glutathione peroxidase, GPx)
(五) 解毒之榖胱甘肽轉移酶(glutathione S-transferase, GST)
(六) 肝胰臟損傷之麩草酸轉胺酶(glutamic oxaloacetic transaminase, GOT)
(七) 肝胰臟損傷之麩丙酮酸轉胺酶(glutamic pyruvic transaminase, GPT)
七、 易感性測試
八、 腸道細菌DNA之萃取
九、 統計分析
(一) 成對樣本t檢定(paired-sample t test)
(二) 卡方獨立性檢定(The chi-squared test of independence)
一、 暴露於APAP和IBU之蝦子經感染實驗後的死亡率
二、 蝦子暴露於不同單一濃度藥物後生理功能基因的轉錄表現
三、 蝦子暴露於不同單一濃度藥物後生理功能之酵素活性的變化
四、 蝦子暴露於APAP和IBU之混合池水後生理功能基因的轉錄表現
五、 蝦子暴露於APAP和IBU混合池水後生理功能之酵素活性的變化
六、 蝦子暴露於單一或混合APAP和IBU池水後腸道菌相的變化
陸、討論
一、 免疫
二、 抗氧化
三、 壓力
四、 解毒
五、 肝胰臟損傷
六、 呼吸
七、 腸道菌相
柒、結論
捌、參考文獻
玖、圖表
附錄


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