本文關(guān)鍵詞： 過氧化甲乙酮 臟器系數(shù) 病理損傷 脂質(zhì)過氧化作用 肝功能　出處：《天津醫(yī)科大學》2013年碩士論文　論文類型：學位論文

【摘要】：MEKP (Methyl Ethyl Ketone Peroxide, MEKP)即過氧化甲乙酮,主要作為玻璃纖維塑料的硬化劑和不飽和聚酯樹脂的固化劑廣泛應用于聚酯及丙烯酸樹脂的生產(chǎn)。目前國內(nèi)MEKP的生產(chǎn)量和使用量逐年在增加,職業(yè)接觸和非職業(yè)接觸人數(shù)不斷增多,MEKP的毒性作用也越來越受到人們的關(guān)注。MEKP具有多方面的毒性效應,主要是對黏膜和皮膚的強烈刺激作用與對各臟器的脂質(zhì)過氧化損害。MEKP的毒性機制可能是通過生成自由基導致脂質(zhì)過氧化反應使細胞膜受損,從而使細胞功能障礙而致凋亡或壞死。國內(nèi)外對于MEKP中毒的病例報告一般是誤食或自殺導致中毒,會導致消化道灼傷,結(jié)構(gòu)和功能均受損。但在工作環(huán)境中,人群通常是經(jīng)呼吸道和皮膚兩種途徑接觸。本研究擬通過大鼠亞慢性吸入MEKP模型,通過MEKP對大鼠機體的慢性損害模式,探討MEKP的毒性機制,為職業(yè)暴露人群的防護和易感人群的篩選提供基礎(chǔ)依據(jù)。 目的 本研究擬通過亞慢性吸入MEKP動物模型,通過MEKP對大鼠機體的慢性損害模式,觀察過氧化甲乙酮亞慢性吸入后對大鼠主要器官的病理學改變,以及對肝臟氧化應激水平的影響,闡述MEKP的脂質(zhì)過氧化損害作用,探討大鼠吸入過氧化甲乙酮(MEKP)氣溶膠后的氧化損傷機制及其職業(yè)人群的醫(yī)學防護提供線索。 方法 建立大鼠MEKP亞慢性染毒模型,SD大鼠100只,雌雄各半,隨機分為5組,每組10只。染毒組暴露于50mg/m3、500mg/m3和1000mg/m3的MEKP氣溶膠,空白對照組暴露于清潔空氣,溶劑對照組暴露于溶劑氣溶膠,6h/d.Sd/w.共13周。 1.染毒期間動物一般情況和病理結(jié)果觀察： 染毒期間,觀察大鼠食欲和活動等一般狀況。染毒結(jié)束后,稱重,CO2麻醉處死解剖大鼠,取肝臟、肺、脾、腎、氣管、卵巢或睪丸,稱重,用體積分數(shù)為10%的中性福爾馬林溶液固定,石蠟包埋,制成5μm厚切片,HE常規(guī)染色,切片,在光學顯微鏡下進行組織病理觀察。 2.大鼠肝臟GSH、MDA含量和SOD活力的測定：用TBA法和考馬斯亮藍染色法分別測定上清液中MDA、GSH及蛋白質(zhì)含量,GSH、MDA含量結(jié)果以"mg/g-pro"表示,用黃嘌呤-黃嘌呤氧化酶-亞硝酸鹽法測定SOD活力,SOD活力結(jié)果以"U/mgpro"表示。 3.大鼠肝臟CYP450、NADH活性的測定：切割標本后,稱取重量。加入一定量的PBS,pH7.4,用手工或勻漿器將標本勻漿充分。離心20分鐘(2000-3000轉(zhuǎn)／分),取一份上清按試劑盒說明操作進行ELISA分析測定樣本活性。 4.大鼠血液中AST、ALT、PLT水平的測定：大鼠CO2麻醉,眼球靜脈取血5mL,加入抗凝劑EDTA,按照AST、ALT、PLT測定試劑盒測定血中AST、ALT、PLT水平。 5.結(jié)果處理：采用SPASS17.0統(tǒng)計分析軟件中的ANOVA方法對試驗數(shù)據(jù)進行差異顯著性檢驗和多重比較,以P0.05為差異有統(tǒng)計學意義。病理組織切片用MotieMC20001.0版顯微照相系統(tǒng)和Adobe photoshop7.0處理。 結(jié)果 1.大鼠臟器系數(shù)變化及主要臟器病理損害情況 大鼠吸入MEKP氣溶膠13周后,1000mg/m3MEKP染毒組雄性大鼠腎臟、胸腺、睪丸臟器系數(shù)顯著低于空白對照組、溶劑對照組及其余各劑量組(P0.05)；1000mg/m3MEKP染毒組雄性大鼠體重顯著低于空白對照組、溶劑對照組。1000mg/m3MEKP染毒組、500mg/m3MEKP染毒組部分大鼠支氣管、肺、肝、脾、腎、睪丸出現(xiàn)明顯損傷,呈現(xiàn)病變發(fā)生率增高及病變程度加重的趨勢。支氣管出現(xiàn)纖毛柱狀上皮脫落,粘膜及粘膜下淋巴細胞浸潤；肺臟損傷主要表現(xiàn)為肺泡過度充氣,肺泡壁毛細血管充血；肝損傷表現(xiàn)為肝細胞及匯管區(qū)小膽管上皮細胞水腫,嗜酸性變及脂肪變性；脾臟腫大,局灶型中性粒細胞浸潤,白髓比例明顯降低。1000mg/m3MEKP染毒組與500mg/m3MEKP染毒組部分雄性大鼠睪丸多級生精細胞發(fā)育不良,精子數(shù)目明顯減少,甚至變性、壞死。 2.大鼠肝臟GSH、MDA含量、SOD活力變化情況 在1000mg/m3MEKP染毒組雄性大鼠中,肝臟內(nèi)GSH含量顯著低于其余各組雄性大鼠肝臟內(nèi)GSH含量,SOD活性顯著低于其余各組雄性大鼠肝臟內(nèi)SOD活力,MDA含量顯著高于空白對照組肝臟內(nèi)MDA含量,差異有統(tǒng)計學意義(P0.05)。在1000mg/m3MEKP染毒組雌性大鼠中,肝臟內(nèi)GSH含量顯著低于其余各組雄性大鼠肝臟內(nèi)GSH含量,SOD活性顯著高于空白對照組、溶劑對照組雌性大鼠肝臟內(nèi)SOD活力,差異有統(tǒng)計學意義P0.05)。在500mg/m3MEKP染毒組,雄性大鼠肝臟內(nèi)GSH含量顯著低于其余各組雄性大鼠肝臟內(nèi)GSH含量,SOD活力顯著低于空白對照組、溶劑對照組雄性大鼠肝臟內(nèi)SOD活性,MDA含量顯著高于空白對照組雄性大鼠肝臟內(nèi)MDA含量,差異有統(tǒng)計學意義(P0.05)；雌性大鼠肝臟內(nèi)SOD活力顯著高于空白對照組、溶劑對照組雌性大鼠肝臟內(nèi)SOD活力,差異有統(tǒng)計學意義P0.05)。實驗結(jié)果表明,大鼠吸入一定劑量的MEKP氣溶膠后,可對肝臟造成一定程度的氧化損傷,MDA含量顯著升高,機體抗氧化能力受損,GSH含量、SOD活性顯著下降。 3.大鼠肝臟NADH與CYP450活性的變化情況 在1000mg/m3MEKP染毒組雄性大鼠,肝臟NADH、CYP450的活性顯著高于空白對照組、溶劑對照組、50mg/m3MEKP染毒組；雌性大鼠肝臟NADH、CYP450的活性顯著高于空白對照組、溶劑對照組,差異有統(tǒng)計學意義(P0.05)；在500mg/m3MEKP染毒組中,雌、雄性大鼠中,肝臟NADH、CYP450的活性顯著高于空白對照組、溶劑對照組、50mg/m3MEKP染毒組,差異有統(tǒng)計學意義(P0.05)；試驗結(jié)果表明大鼠吸入MEKP后,MEKP可結(jié)合并破壞大鼠肝臟內(nèi)的NADH與CYP450,使其活性降低,繼而導致活性下降。 4.大鼠血液中AST、ALT、PLT水平變化情況 在1000mg/m3MEKP染毒組中,雄性大鼠血液中AST測定結(jié)果顯著高于空白對照組、溶劑對照組、50mg/m3MEKP染毒組,雌性大鼠血液中AST測定結(jié)果顯著高于空白對照組、溶劑對照組、50mg/m3MEKP染毒組、500mg/m3MEKP染毒組,雄性大鼠血液中ALT測定結(jié)果顯著高于空白對照組、溶劑對照組、50mg/m3MEKP染毒組,雌性大鼠肝臟ALT測定結(jié)果顯著高于空白對照組、溶劑對照組、50mg/m3MEKP染毒組,差異有統(tǒng)計學意義(P＜0.05)。500mg/m3MEKP染毒組中,雌雄大鼠血液中AST測定結(jié)果顯著高于空白對照組、溶劑對照組、50mg/m3MEKP染毒組,差異有統(tǒng)計學意義(P0.05)；大鼠吸入MEKP氣溶膠13周后,肝臟匯管區(qū)細胞出現(xiàn)脂肪滴,發(fā)生脂肪變性,纖維化病變,導致肝功能異常。1000mg/m3MEKP染毒組雄性大鼠血小板水平與空白對照組、溶劑對照組、50mg/m3MEKP染毒組之間相比,出現(xiàn)顯著減少,差異有統(tǒng)計學意義(P0.05)。 結(jié)論 1.500mg/m3濃度的MEKP氣溶膠可對大鼠臟器產(chǎn)生明顯的病理損傷作用,雄性大鼠的損傷情況相比雌性大鼠較為嚴重。提示MEKP對大鼠的損傷作用有性別差異,對雄性大鼠造成的損傷作用較嚴重。 2.500mg/m3濃度的MEKP氣溶膠可以使大鼠肝臟氧化應激水平顯著下降,MDA含量升高,GSH含量、SOD活性顯著下降,說明MEKP在機體內(nèi)產(chǎn)生了過多的自由基。 3.500mg/m3濃度的MEKP氣溶膠會導致大鼠肝臟功能嚴重受損,AST、ALT升高、PTL減少,肝臟內(nèi)NADH、CYP450活性顯著降低,提示通過脂質(zhì)過氧化損傷作用導致肝損傷。
[Abstract]:MEKP ( Methyl Ethyl Ester Peroxide , MEKP ) , i.e . methyl ethyl ketone peroxide , is widely used in the production of polyester and acrylic resin . Purpose In this study , the effects of MEKP on the pathological changes of main organs of rats and the effect of MEKP on the oxidative stress level in rats were investigated by subchronic inhalation of MEKP animal model . The mechanism of oxidative damage after inhalation of methyl ethyl ketone peroxide ( MEKP ) aerosol and medical protection of the occupational population were discussed . method The rats were divided into 5 groups randomly divided into 5 groups . The exposed groups were exposed to 50 mg / m3 , 500 mg / m3 and 1000 mg / m3 of MEKP aerosol . The blank control group was exposed to clean air , and the solvent control group was exposed to solvent aerosol , 6 h / d . sd / w for 13 weeks . 1 . Observation of the general and pathological results of the animals during the exposure period : During the exposure period , the general conditions of appetite and activity were observed in rats . After the end of exposure , the rats were sacrificed by weight , CO2 anesthesia , and the liver , lung , spleen , kidney , trachea , ovary or testis were weighed . 2 . The content of GSH , MDA and SOD in the liver of rats were determined : the content of MDA , GSH , protein , GSH and MDA in the supernatant were determined by TBA method and Coma brilliant blue staining respectively . The results of GSH and MDA content were expressed as " mg / g - pro " . The activity of SOD was determined by xanthine - xanthine oxidase - nitrite method , and the results of SOD activity were expressed as " U / mgpro " . 3 . Determination of CYP450 and NADH activity in rat liver : After cutting the specimen , weigh it . Add a certain amount of PBS , pH 7.4 , and use manual or homogenizer to make the sample homogenate adequate . After centrifugation for 20 minutes ( 2000 - 3000 rpm ) , take a supernatant and make ELISA analysis and assay sample activity according to the instructions of the kit . 4 . Determination of AST , ALT and PLT in blood of rats : CO2 anesthesia in rats , 5 mL of blood from eyeball vein , anticoagulant EDTA , AST , ALT and PLT levels were determined by AST , ALT and PLT measurement kits . 5 . Results : The statistical significance of ANOVA in SPAS S17 . 0 statistical analysis software was statistically significant for the test data . The pathological tissue sections were treated with Motif MC20001 . 0 micrographic system and Adobe Photoshop 7.0 . Results 1 . Changes of organ coefficients and pathological damage of major organs in rats After inhalation of MEKP aerosol for 13 weeks in rats , the rat kidney , thymus and testis were significantly lower than those in the control group , the solvent control group and the rest groups ( P0.05 ) . 2 . Changes of GSH , MDA and SOD activity in rat liver The content of GSH in liver was significantly lower than that of the other groups ( P0.05 ) . The content of GSH in liver was significantly lower than that in the control group ( P0.05 ) . The content of GSH in liver of male rats was significantly lower than that in the control group ( P0.05 ) . The SOD activity in liver of female rats was significantly higher than that in the blank control group ( P0.05 ) . The experimental results showed that after inhalation of certain dose of MEKP aerosol in rats , some degree of oxidative damage to the liver could be caused , the content of MDA increased significantly , the anti - oxidation ability of the organism was damaged , the GSH content and SOD activity were significantly decreased . 3 . Changes of hepatic NADH and CYP450 activity in rats The activity of NADH and CYP450 in liver NADH and CYP450 was significantly higher than that in blank control group , solvent control group and 50mg / m3MEKP group . The activity of NADH and CYP450 in liver of female rats was significantly higher than that in control group , solvent control group and 50mg / m3MEKP group . 4 . Changes of AST , ALT and PLT in Blood of Rats The results of AST determination in blood of male rats were significantly higher than those in control group , solvent control group , 50mg / m3MEKP group and 500 mg / m3MEKP group . Conclusion The effects of MEKP on the injury of male rats were more serious than those in female rats . 2 . The concentration of MEKP in the concentration of 500 mg / m3 could significantly decrease the level of oxidative stress in rat liver , the content of MDA increased , the content of GSH increased , the activity of SOD decreased significantly , indicating that MEKP produced too many free radicals in the organism . 3.500 mg / m ~ 3 concentration of MEKP aerosol can cause severe hepatic injury , AST , ALT increase , decrease of liver function , decrease of NADH and CYP450 activity in liver , suggesting that liver injury can be caused by lipid peroxidation damage .

【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R114

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