Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20837
標題: 建立酒精性肝病與奈米顆粒引發肺傷害之動物模式與其相關研究
To establish the animal models for alcohol liver disease and nanoparticles-induced lung injuries in rat/mice and related researches
作者: 林煒翔
Lin, Wei-Hsiang
關鍵字: Alcohol
酒精
Catechin alcohol
Acute liver injury
Chronic liver injury
Reactive oxygen species
Nanoparticles
ultra-fine TiO2
single-wall carbon nanotubes
placenta growth factor
microarray
pulmonary emphysema
茶多酚
急性肝損傷
慢性肝損傷
自由基
巨噬細胞
生物醫學
性傷害
肺氣腫
脂肪酸
奈米粒子
超微細二氧化鈦
單層奈米碳管
基因微陣列
胎盤生長因子
肺氣腫
出版社: 生命科學系
摘要: 摘 要(第一部份 酒精添加茶多酚對酒精性肝病之效應) 肝臟是人體負責解毒或代謝作用重要器官。而酗酒與肝炎病毒感染是造成肝病的兩個主要病因,隨著全球酒精消費量逐年增加,也使得酒精性肝病(alcohol liver disease)的發病率明顯上升,其中機轉與活性氧的產生有密切關係,因此找尋預防或減緩酒精性肝損傷的抗氧化食品為營養醫學重要議題。本研究評估藉由在酒精中添加茶多酚(茶酒)之抗氧化能力與在預防急性或慢性肝損傷之作用。首先本研究發現茶酒明顯的較單純酒精具有較好之抗O2-‧、H2O2及HOCl之能力。再將實驗分為兩大部份,第一部分為急性肝損傷,分別利用36%酒精及茶酒,進行股靜脈注射處理五分鐘(0.25ml/250g)後,再以每小時0.12ml/250g的速度,連續注射六個小時,觀察大白鼠於處理1-6小時後,活體肝臟表面自由基及第六個小時的離體自由基(血液及膽汁);第二部分為慢性肝損傷,將實驗動物分別餵食正常飼料、高脂食物組、高脂食物加上36%酒精灌胃以及高脂食物加上茶酒灌胃,餵養一個月誘導肝損傷,再進行生化指標、膽汁自由基、切片染色及西方點墨法等分析。急性肝損傷結果顯示,連續注射36%酒精6小時後,其血液、膽汁及肝臟活體自由基的上升;而連續注射茶酒的組別,與酒精組相比則能降低血液、膽汁和肝臟表面自由基。在慢性肝損傷方面,餵食高脂食物加上36%酒精灌胃的組別,血液中脂肪(TG、T-CHO、VLDL及LDL)的濃度上升、膽汁自由基增加、肝臟中自由基所造成的醛類(4-hydroxynonenal/4HNE)堆積含量上升、ED-1 postive的Kupffer cells增多及酒精性脂肪肝明顯表現;而高脂食物加上茶酒灌胃的組別則能降低:血液中脂肪(TG、T-CHO、VLDL及LDL)的濃度、膽汁自由基、肝臟中自由基所造成的醛類(4-hydroxynonenal/4HNE)堆積含量、ED-1 postive的Kupffer cells及酒精性脂肪肝,並提高血液中好的脂蛋白(HDL)的含量。以西方點墨法分析,發現高脂食物加上茶酒灌胃的組別會增加抗氧化蛋白(CuZn-SOD/CuZn-superoxide dismutase)與抗細胞凋亡蛋白質(bcl-2)的表現量,同時也會降低促細胞凋亡蛋白(bax)及調控脂肪酸合成蛋白(SREBP-1) 的表現量。綜合以上結果,茶酒較36%酒精具有較好之抗O2-‧、H2O2及HOCl之能力。長期給予茶酒,可以提升抗氧化蛋白的表現,對急性或慢性肝損傷具有保護作用,並降低細胞凋亡,及脂肪酸合成。這些保護機轉可以減緩酒精所造成肝損傷。 摘 要(第二部份 吸入奈米顆粒(超微細二氧化鈦和單層奈米碳管)造成小鼠肺毒性之研究) 在最近幾年裡,奈米科技在科學、工業以及生物醫學都有重要的應用,但是這項科技的使用是否對我們和環境是安全的?其答案仍是不明確的。事實上已有許多研究顯示,在幾種動物模式中,奈米粒子會造成很顯著的吸入性傷害,如先前研究指出超微細二氧化鈦(ultrafine/ UF-TiO2)、奈米碳管和煤礦粉塵可導致大鼠肺部慢性發炎、纖維化和肺癌,且UF-TiO2會活化巨噬細胞的功能,導致持續性的發炎反應,使之產生大量的氧自由基及一些生長調控蛋白,而吸入這些奈米粒子會比微米粒子產生更顯著的肺部毒性。吸入奈米粒子能導致肺毒性,然而目前探討奈米粒子引發小鼠肺部病變之機轉的研究,卻是相當缺乏的。在本研究中我們利用超微細二氧化鈦(UF-TiO2)和單層奈米碳管(Single-Wall Carbon Nanotubes, SWCNT)來研究奈米粒子造成小鼠肺部毒性與其病變的機轉。小鼠的肺部將以氣管內管的方式,分別注入0.1或0.5 毫克的Fine/F-TiO2 R type、UF-TiO2 R type或SWCNT處裡三天及七天。我們發現UF-TiO2 R type與SWCNT會導致小鼠產生上皮狀肉芽腫(granulomas)、肺泡間隔被破壞的肺氣腫(pulmonary emphysema)病理現象、第二型上皮細胞(type II pneumocyte)增生、肺泡的巨噬細胞浸潤、肺部細胞增生及肺泡間隔細胞凋亡。我們也利用cDNA基因微陣列(microarray)及Affymetrix GeneChip等方法,偵測UF-TiO2處理小鼠肺臟所產生的基因,我們發現UF-TiO2 particles會誘導小鼠肺臟所產生數以百計的有差異基因表現,其中包括一些細胞激素(cytokines)、化學激素(chemokines)如vEGFc、PlGF、Flt-3、MIP-1、MIP-2、MCP-1、IL-1、C-C和C-X-C,及誘導一些調控細胞週期物質(cyclin A、B和cdk2a)的產生。最後我們將結果經由KEGG和BioCata等資料庫比對,並進行分析。根據基因微陣列的結果及路徑分析,我們找出胎盤生長因子(placenta growth factor/PlGF)及其他chemokines、cell cycle、apoptosis或complement pathway都可能參與UF-TiO2 R type所引起之肺毒性的病變機轉。總結:我們的研究不僅設計一個動物模式,提供奈米粒子於活體中對肺部毒性的驗證,也指出了奈米粒子可以導致肺部損傷及其可能的致病路徑。
Abstract(Part I. The effects of catechin alcohol on alcohol liver disease) Liver is an important detoxifying and metabolic organ in the human body. Drinking and viral infection are two of the main causes of liver diseases. Obvious correlation was observed between the increasing global alcohol consumption and the rising incidence of the alcohol liver diseases. Aumulating evidence indicated that reactive oxygen species (ROS) contribute to alcohol-induced liver diseases. Therefore, We test whether adding antioxidant ingredients, like catechins, in the 36% alcohol may prevent or reduce alcohol liver damage. Our results showed that catechin alcohol can significantly scavenge O2-‧, H2O2 and HOCl in vitro. Furthermore, in our acute liver damage animal model, catechin alcohol (0.25 ml/250g) reduced the value of ROS from the liver surface and bile and blood. We also found that the occurrence of chronic alcoholic liver disease was significantly reduced after one month of oral catechin alcohol when compared to 36% alcohol. We found the levels of blood, bile, and liver ROS were reduced in the catechin alcohol treated high fat-feeding rats, also , the level of VLDL and LDL in blood, the contents of 4- hydroxynonenal/4HNE in liver and the number of Kupffer cells (ED-1 positive stains) decreased and sparse alcohol fatty liver were observed.. Western blot analysis showed an up-regulation of CuZn-superoxide dismutase (CuZn-SOD) and Bcl-2 protein and a down-regulation of Bax and SREBP-1 protein in the catechin alcohol treated livers. In conclusion, catechin alcohol with antioxidant O2-‧,H2O2 and HOCl offers a protection against alcohol induced oxidative injury via the up-regulation of anti-apoptotic and antioxidant proteins. Abstract(Prat II. To Study the Pulmonary Toxicity of Nano-Particles(UF-TiO2 and Single-Wall Carbon Nanotubes) in mice after intratracheal instillation.) Nanotechnology has important scientific, industrial, and bio-medical applications,however, its effects on human health and and environmental safetyare still uncertain. In fact, a lot of studies have suggested that nano-particle inhalation might induce pulmonary injuries in several animal models. Previous studies have shown that ultra fine-titanium dioxide (UF-TiO2), carbon nanotube, and colliery dust might induce chronic pulmonary inflammation, pulmonary fibrosis, and lung cancer in rat . The macrophages thatactivated by UF-TiO2produce lots of oxygen free radicals and some growth factors, that lead to chronic inflammation. Inhalation of nanoparticles could induce more severe pulmonary toxicity than microparticles. Nanoparticle inhalation can induce pulmonary toxicity, however its pathogenic mechanism is not fully understood. In this study, UF-TiO2 R type and Single-Wall Carbon Nanotubes (SWCNT) were used to investigate their pulmonary toxicity and molecular mechanisms of pathogenesis . Mice were intratracheally instilled with 0.1 or 0.5 mg of fine-TiO2 R type (F-TiO2 R type), UF-TiO2 R type and SWCNT in 3 days or 7 days. UF-TiO2 R type and SWCNT induced epithelioid granulomas, pulmonary emphysema with extensive disruption of alveolar septa, type II pneumocyte hyperplasia, foamy macrophage accumulation, pulmonary cell proliferation and alveolar septa cells apoptosis. We also utilize cDNA microarray and Affymetrix GeneChip to detect the gene expression profiles of mice's lung with UF-TiO2 R type treatment. We found that hundreds of distinct genes were differentially expressed, including cytokines, chemokines (vEGFc, PlGF, Flt-3, MIP-1, MIP-2, MCP-1, IL-1, C-C and C-X-C), and cell cycle regulators (cdk2, cyclin A, and B).Furthermore, We used the KEGG and BioCata databases to address how these genes might involve in pulmonary pathogenesis.. According to the arrays' data and pathway analysis, we found that placenta growth factor (PlGF), cell cycle, apoptosis, chemokine and complement pathways might involve in UF-TiO2 R type-induced pulmonary pathological changes. In conclusion, our study has not only pointed out that nano-particle could induce pulmonary injury, but also setup a platform of animal model to examine the pulmonary toxicity of nanoparticles in vivo. We also provide possible pathogenic pathways that might be involved.
URI: http://hdl.handle.net/11455/20837
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