大鼠吸入过氧化甲乙酮后脂质过氧化损伤作用实验研究

发布时间：2018-02-01 03:21

本文关键词： 过氧化甲乙酮脏器系数病理损伤脂质过氧化作用肝功能　出处：《天津医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：MEKP (Methyl Ethyl Ketone Peroxide, MEKP)即过氧化甲乙酮,主要作为玻璃纤维塑料的硬化剂和不饱和聚酯树脂的固化剂广泛应用于聚酯及丙烯酸树脂的生产。目前国内MEKP的生产量和使用量逐年在增加,职业接触和非职业接触人数不断增多,MEKP的毒性作用也越来越受到人们的关注。MEKP具有多方面的毒性效应,主要是对黏膜和皮肤的强烈刺激作用与对各脏器的脂质过氧化损害。MEKP的毒性机制可能是通过生成自由基导致脂质过氧化反应使细胞膜受损,从而使细胞功能障碍而致凋亡或坏死。国内外对于MEKP中毒的病例报告一般是误食或自杀导致中毒,会导致消化道灼伤,结构和功能均受损。但在工作环境中,人群通常是经呼吸道和皮肤两种途径接触。本研究拟通过大鼠亚慢性吸入MEKP模型,通过MEKP对大鼠机体的慢性损害模式,探讨MEKP的毒性机制,为职业暴露人群的防护和易感人群的筛选提供基础依据。目的本研究拟通过亚慢性吸入MEKP动物模型,通过MEKP对大鼠机体的慢性损害模式,观察过氧化甲乙酮亚慢性吸入后对大鼠主要器官的病理学改变,以及对肝脏氧化应激水平的影响,阐述MEKP的脂质过氧化损害作用,探讨大鼠吸入过氧化甲乙酮(MEKP)气溶胶后的氧化损伤机制及其职业人群的医学防护提供线索。方法建立大鼠MEKP亚慢性染毒模型,SD大鼠100只,雌雄各半,随机分为5组,每组10只。染毒组暴露于50mg/m3、500mg/m3和1000mg/m3的MEKP气溶胶,空白对照组暴露于清洁空气,溶剂对照组暴露于溶剂气溶胶,6h/d.Sd/w.共13周。 1.染毒期间动物一般情况和病理结果观察：染毒期间,观察大鼠食欲和活动等一般状况。染毒结束后,称重,CO2麻醉处死解剖大鼠,取肝脏、肺、脾、肾、气管、卵巢或睾丸,称重,用体积分数为10%的中性福尔马林溶液固定,石蜡包埋,制成5μm厚切片,HE常规染色,切片,在光学显微镜下进行组织病理观察。 2.大鼠肝脏GSH、MDA含量和SOD活力的测定：用TBA法和考马斯亮蓝染色法分别测定上清液中MDA、GSH及蛋白质含量,GSH、MDA含量结果以"mg/g-pro"表示,用黄嘌呤-黄嘌呤氧化酶-亚硝酸盐法测定SOD活力,SOD活力结果以"U/mgpro"表示。 3.大鼠肝脏CYP450、NADH活性的测定：切割标本后,称取重量。加入一定量的PBS,pH7.4,用手工或匀浆器将标本匀浆充分。离心20分钟(2000-3000转／分),取一份上清按试剂盒说明操作进行ELISA分析测定样本活性。 4.大鼠血液中AST、ALT、PLT水平的测定：大鼠CO2麻醉,眼球静脉取血5mL,加入抗凝剂EDTA,按照AST、ALT、PLT测定试剂盒测定血中AST、ALT、PLT水平。 5.结果处理：采用SPASS17.0统计分析软件中的ANOVA方法对试验数据进行差异显著性检验和多重比较,以P0.05为差异有统计学意义。病理组织切片用MotieMC20001.0版显微照相系统和Adobe photoshop7.0处理。结果 1.大鼠脏器系数变化及主要脏器病理损害情况大鼠吸入MEKP气溶胶13周后,1000mg/m3MEKP染毒组雄性大鼠肾脏、胸腺、睾丸脏器系数显著低于空白对照组、溶剂对照组及其余各剂量组(P0.05)；1000mg/m3MEKP染毒组雄性大鼠体重显著低于空白对照组、溶剂对照组。1000mg/m3MEKP染毒组、500mg/m3MEKP染毒组部分大鼠支气管、肺、肝、脾、肾、睾丸出现明显损伤,呈现病变发生率增高及病变程度加重的趋势。支气管出现纤毛柱状上皮脱落,粘膜及粘膜下淋巴细胞浸润；肺脏损伤主要表现为肺泡过度充气,肺泡壁毛细血管充血；肝损伤表现为肝细胞及汇管区小胆管上皮细胞水肿,嗜酸性变及脂肪变性；脾脏肿大,局灶型中性粒细胞浸润,白髓比例明显降低。1000mg/m3MEKP染毒组与500mg/m3MEKP染毒组部分雄性大鼠睾丸多级生精细胞发育不良,精子数目明显减少,甚至变性、坏死。 2.大鼠肝脏GSH、MDA含量、SOD活力变化情况在1000mg/m3MEKP染毒组雄性大鼠中,肝脏内GSH含量显著低于其余各组雄性大鼠肝脏内GSH含量,SOD活性显著低于其余各组雄性大鼠肝脏内SOD活力,MDA含量显著高于空白对照组肝脏内MDA含量,差异有统计学意义(P0.05)。在1000mg/m3MEKP染毒组雌性大鼠中,肝脏内GSH含量显著低于其余各组雄性大鼠肝脏内GSH含量,SOD活性显著高于空白对照组、溶剂对照组雌性大鼠肝脏内SOD活力,差异有统计学意义P0.05)。在500mg/m3MEKP染毒组,雄性大鼠肝脏内GSH含量显著低于其余各组雄性大鼠肝脏内GSH含量,SOD活力显著低于空白对照组、溶剂对照组雄性大鼠肝脏内SOD活性,MDA含量显著高于空白对照组雄性大鼠肝脏内MDA含量,差异有统计学意义(P0.05)；雌性大鼠肝脏内SOD活力显著高于空白对照组、溶剂对照组雌性大鼠肝脏内SOD活力,差异有统计学意义P0.05)。实验结果表明,大鼠吸入一定剂量的MEKP气溶胶后,可对肝脏造成一定程度的氧化损伤,MDA含量显著升高,机体抗氧化能力受损,GSH含量、SOD活性显著下降。 3.大鼠肝脏NADH与CYP450活性的变化情况在1000mg/m3MEKP染毒组雄性大鼠,肝脏NADH、CYP450的活性显著高于空白对照组、溶剂对照组、50mg/m3MEKP染毒组；雌性大鼠肝脏NADH、CYP450的活性显著高于空白对照组、溶剂对照组,差异有统计学意义(P0.05)；在500mg/m3MEKP染毒组中,雌、雄性大鼠中,肝脏NADH、CYP450的活性显著高于空白对照组、溶剂对照组、50mg/m3MEKP染毒组,差异有统计学意义(P0.05)；试验结果表明大鼠吸入MEKP后,MEKP可结合并破坏大鼠肝脏内的NADH与CYP450,使其活性降低,继而导致活性下降。 4.大鼠血液中AST、ALT、PLT水平变化情况在1000mg/m3MEKP染毒组中,雄性大鼠血液中AST测定结果显著高于空白对照组、溶剂对照组、50mg/m3MEKP染毒组,雌性大鼠血液中AST测定结果显著高于空白对照组、溶剂对照组、50mg/m3MEKP染毒组、500mg/m3MEKP染毒组,雄性大鼠血液中ALT测定结果显著高于空白对照组、溶剂对照组、50mg/m3MEKP染毒组,雌性大鼠肝脏ALT测定结果显著高于空白对照组、溶剂对照组、50mg/m3MEKP染毒组,差异有统计学意义(P＜0.05)。500mg/m3MEKP染毒组中,雌雄大鼠血液中AST测定结果显著高于空白对照组、溶剂对照组、50mg/m3MEKP染毒组,差异有统计学意义(P0.05)；大鼠吸入MEKP气溶胶13周后,肝脏汇管区细胞出现脂肪滴,发生脂肪变性,纤维化病变,导致肝功能异常。1000mg/m3MEKP染毒组雄性大鼠血小板水平与空白对照组、溶剂对照组、50mg/m3MEKP染毒组之间相比,出现显著减少,差异有统计学意义(P0.05)。结论 1.500mg/m3浓度的MEKP气溶胶可对大鼠脏器产生明显的病理损伤作用,雄性大鼠的损伤情况相比雌性大鼠较为严重。提示MEKP对大鼠的损伤作用有性别差异,对雄性大鼠造成的损伤作用较严重。 2.500mg/m3浓度的MEKP气溶胶可以使大鼠肝脏氧化应激水平显著下降,MDA含量升高,GSH含量、SOD活性显著下降,说明MEKP在机体内产生了过多的自由基。 3.500mg/m3浓度的MEKP气溶胶会导致大鼠肝脏功能严重受损,AST、ALT升高、PTL减少,肝脏内NADH、CYP450活性显著降低,提示通过脂质过氧化损伤作用导致肝损伤。
[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 .

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

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