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標題: | 應用質譜儀為基礎的脂質體學探討萘對小鼠的初期致毒機制 Mass Spectrometry-Based Lipidomics to Study Early Event of Naphthalene Toxicity in Mice |
作者: | Ke-Han Chen 陳科翰 |
指導教授: | 林靖愉 |
關鍵字: | ?,磷脂醯膽鹼,脂質體學,初期時間點,肺部毒性, Naphthalene,Phosphatidylcholines (PCs),Lipidomics,Early time point,Lung toxicity, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 萘為結構最簡單的多環芳香烴,經不完全燃燒或含萘產品揮發等過程排放至環境中,又IARC將萘歸類為2B可能致癌物質,因此,萘對生物體造成的健康影響不容忽視。過去組織病理學研究得知萘對小鼠呼吸道產生選擇性的細胞傷害,不僅24小時暴露萘後對非纖毛細胞 (Clara cell)產生傷害,萘毒性在初期 (6小時內)對非纖毛細胞也已經產生傷害。細胞型態學變化可能與細胞膜成分的擾動相關,脂質為構成細胞膜重要的成分,探討脂質受萘影響以了解萘的致毒機制是需要的。因此,本研究針對細胞膜中重要的脂質-磷脂醯膽鹼 (Phosphatidylcholines)來探討萘的初期致毒機制以解釋細胞傷害。
本實驗將小鼠經腹腔注射200 mg/kg萘後1, 2, 3, 6小時犧牲,同時在0小時施打橄欖油做為對照組。利用蘇木精-伊紅染色法 (Hematoxylin and Eosin staining)與免疫組織化學法 (Immunohistochemistry)等方法觀察小鼠肺臟細胞型態學的變化。另一方面,收集肺臟、肝臟及血清樣本並以極致液相層析儀搭配串聯式質譜儀 (UPLC-MS/MS)分析各組織及血清的脂質體變化,經數據前處理後利用及偏最小平方判別分析 (Partial least squared discriminate analysis)觀察不同組別分群結果,最後,應用Kruskal-Wallis以及Mann-Whitney的事後檢定方法來找出不同組別間顯著差異的磷脂醯膽鹼。 組織病理學結果得知非纖毛細胞於暴露萘2小時後開始造成細胞腫脹與空泡化,當暴露萘3小時後非纖毛細胞產生囊泡,暴露萘6小時後,除了持續的細胞腫脹與空泡化,些許的非纖毛細胞開始從支氣管上剝落。小鼠肺臟減少monounsaturated phosphatidylcholines可能與細胞型態的變化有關;減少saturated phosphatidylcholines與polyunsaturated phosphatidylcholines將提高細胞膜的通透性。另一方面,小鼠暴露萘1、2、6小時後肺臟lyso-phosphatidylcholines 降低與減緩細胞通透性相關,且增加polyunsaturated plasmanylcholines也與減緩細胞通透性相關。小鼠肝臟plamenycholines增加將提高自我防禦的機制,且擁有許多抗氧化的脂肪酸,如:亞麻油酸 (Linoleic acid)與花生四烯酸 (Arachidonic acid),使小鼠肝臟較能提高自我保護能力。3小時萘暴露組發現小鼠肺臟中大部分的磷脂醯膽鹼與控制組相比後沒有太多顯著變化,此結果與短暫的修復機制有關。最後,小鼠肺臟與血清中PC(16:0/18:1)有一致的變化情形,該脂質將有潛力成為重要的生物指標,未來需要再進行諸多實驗來驗證此生物指標的真實性。 本研究顯示在初期暴露萘後對小鼠肺臟與肝臟的磷脂醯膽鹼已產生擾動,小鼠肺臟於3小時萘暴露後產生短暫的修復機制,小鼠肺臟的磷脂醯膽鹼改變可能造成細胞型態變化,而小鼠肝臟含有許多抗氧化的脂肪酸,此差異可能是使肺臟在暴露萘後成為一個高感受的標的器官的原因。 Naphthalene is the simplest polycyclic aromatic hydrocarbon and widely exists in the environment. Previous studies have revealed that naphthalene-induced airway non-ciliated (Clara) cell injuries in mice at earlier time points. Since the changes of cell morphology may be greatly related to membrane components, it is critical to understand molecular events in the development of naphthalene toxicity in lipid level. Currently, we focus on the changes of the most abundant membrane lipids, phos- phatidylcholines to characterize naphthalene toxicity. Male ICR mice were treated with 200 mg/kg naphthalene by intraperitoneally (i.p.) for 1, 2, 3 or 6 hours. Hematoxylin and eosin staining and immunohisto- chemistry were used to characterize cellular morphological changes in the lung. The lung, liver tissues and serum were collected and prepared for mass spectrometry (MS) based lipidomic analysis. Phosphatidylcholines were analyzed by ultra performance liquid chromatography coupled with tandem mass spectrometer (UPLC-MS/MS). After pre-processing MS data, multivariable analysis, such as partial least squared discriminate analysis was used to examine phosphatidylcholine variations among samples. Kruskal-Wallis and Mann-Whitney as the post hoc were used in finding the significant changes of phosphatidylcholines among different groups. The histopathologic results showed Clara cells were injured as early as 2 hours post dose. As time period increased, Clara cell had apical blebs and more severe injury at 3 and 6 hour treated groups. Decreased monounsaturated phosphatidyl- cholines in mouse lung might relate to cell morphologic changes, especially for 6 hour treated group. Decreased saturated and polyunsaturated phosphatidylcholines in the mouse lung after naphthalene treatment was related to changes of membrane rigidity, flexibility and fluidity which lead to membrane permeability. On the other hand, decreased lyso-phosphatidylcholines among 1, 2 and 6 hour treated groups and increased polyunsaturated plasmanylcholines in the mouse lung might be related with resistant to membrane permeability. Plamenycholines in mouse liver were significantly increased to add the ability to fight with reactive oxidative species. Moreover, numerous distinct fatty acids, such as linoleic acid and arachidonic acid in the mouse liver protect the liver from naphthalene-induced oxidative stress. Interestingly, several phosphatidylcholines were not changed compared with control group in 3 hour treated group in the mouse lung. This may relate to a short-term repair mechanism. Finally, a consistent trend of the change of PC(16:0/18:1) in the lung and serum might serve as a potential biomarker for Clara cell injury. Further tests to confirm our finding are needed. In conclusion, the perturbations of phosphatidylcholines were found at very early time points of naphthalene intervention in the mouse lung and liver. Changes of phosphatidylcholines are related with changes of cell morphology in the lung. Limited changes of phosphatidylcholines at 3 hours in mouse lung after naphthalene intervention may be relate to short-term repair. Distinct fatty acids in the target (lung) and non-target (liver) may partially explain organ-specific susceptibility. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52175 |
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