小鼠急性肝损伤模型的建立及GSTA1分析

发布时间：2018-06-22 13:28

本文选题：急性肝损伤 + 四氯化碳　；参考：《东北农业大学》2012年硕士论文

【摘要】：近年来肝脏疾病对人类的健康和社会的发展造成严重威胁,养殖业中肝病的发病率明显增高,不仅给养殖业造成严重的经济损失,而且通过食物链直接或间接对人类健康造成危害,引发诸多食品安全问题。急性肝损伤是多种肝脏疾病的发生、发展及最终走向肝功能衰竭的始动环节和共同途径,建立与人类急性肝损伤发展病变过程相似的动物模型极为重要。深入研究和探讨急性肝损伤的发病机制以及治疗措施,对慢性肝脏疾病的防治具有重要意义。GSTA1是Ⅱ相结合反应同功酶GST的一个亚型,在抗氧化损伤、抗肿瘤方面具有重要作用,并有可能成为药物研发重要的新方向。目前关于GSTA1在急性肝损伤中的变化研究较少,尚未见GSTA1在急性肝损伤中的作用研究。明确GSTA1在急性肝损伤中的变化将为GSTA1保护作用及其机制的研究提供理论基础和试验依据。选用雄性昆明小鼠为实验动物,研究并明确四氯化碳(CCl4)致小鼠急性化学性肝损伤、醋氨酚(APAP)致小鼠急性药物性肝损伤、乙醇致小鼠急性酒精性肝损伤的建立方法和评价方法。通过筛选获得最佳的染毒途径、剂量和时间,以此为基础复制三种小鼠急性肝损伤模型。CCl4模型以血清转氨酶(ALT、AST)、肝组织指标(SOD、MDA、GSH、GSH-Px)和肝脏病理组织学分析进行评价；APAP模型以血清转氨酶(ALT、AST)、肝组织指标(SOD、MDA、GSH、GSH-Px、NO)和肝脏病理组织学分析进行评价；乙醇模型以血清转氨酶(ALT、 AST)、肝组织指标(SOD、MDA、GSH、GSH-Px、TG)和肝脏病理组织学分析进行评价。并在成功复制的各模型中,检测血清和肝脏中GSTA1含量和活性变化情况。GSTA1含量检测为ELISA原理,采用小鼠GSTA1试剂盒进行。GSTA1活性检测采用比色法,以NBD-Cl为底物,通过GSTA1所催化的酶促反应生成物NBD-SG的吸光度来计算。血清中酶活性表达为每分钟每毫升血清中GSH反应生成物的量,肝组织中酶活性表达为每分钟每毫克胞浆蛋白中GSH反应生成物的量。研究结果表明： 1.以0.35%CCl4油溶液,按10mL·kg-1剂量灌胃,染毒24h可成功复制CC14致小鼠急性肝损伤模型。模型组血清转氨酶活性升高,与对照组相比差异极显著(P0.01),肝组织中各指标变化与对照组相比均差异极显著(P0.01),病理切片观察到模型组小鼠肝细胞颗粒变性,胞浆崩解,细胞核浓缩,炎性细胞浸润。血清中GSTA1含量和活性显著增加(P0.01),而肝脏中GSTA1含量和活性均显著降低(P0.01)。 2.以200mg·kg-1剂量APAP灌胃,染毒12h可成功复制小鼠急性肝损伤模型。模型组血清转氨酶活性升高,与对照组相比差异极显著(P0.01),肝组织中各指标变化与对照组相比均差异极显著(P0.01),病理切片观察到模型组肝组织充血、淤血,炎性细胞浸润,肝索模糊,胞核固缩。血清中GSTA1含量和活性显著增加(P0.01),而肝脏中GSTA1含量和活性均显著降低(P0.01)。 3.以50%乙醇,按14mL·kg-1剂量灌胃,染毒8h可成功复制小鼠急性肝损伤模型。模型组血清转氨酶活性升高,与对照组相比差异极显著(P0.01),肝组织中各指标变化与对照组相比均差异极显著(P0.01),病理切片观察到模型组小鼠肝组织有圆形空泡、炎性细胞浸润,肝细胞点状坏死。血清中GSTA1含量和活性显著增加(P0.01),而肝脏中GSTA1含量和活性均显著降低(P0.01)。本试验成功复制急性化学性、药物性、酒精性肝损伤动物模型。在三种急性肝损伤模型中,GSTA1含量和活性在血清中显著增加(P0.01),且在肝组织中显著降低(P0.01),表明急性肝损伤时GSTA1由肝脏释放入血,参与机体抗氧化活动,从而起到保护肝脏的作用。血清中GSTA1的变化表明GSTA1对于急性肝损伤的评价具有重要意义。本研究为继续探索保肝药物对GSTA1调节作用和GSTA1保护肝脏的分子机制打下坚实基础并提供可靠理论数据。
[Abstract]:In recent years , liver diseases have posed a serious threat to human health and social development , and the incidence of liver diseases in the breeding industry is significantly increased . It is very important not only to cause serious economic losses to the breeding industry , but also to directly or indirectly harm the human health through the food chain .

The acute hepatic injury induced by acute chemical liver injury and alcohol induced acute alcoholic liver injury in mice induced by carbon tetrachloride ( CCl _ 4 ) induced acute hepatic injury in mice was studied by using male Kunming mice as experimental animals .
APAP model was evaluated by serum transaminase ( ALT , AST ) , liver tissue index ( SOD , MDA , GSH , GSH - Px , NO ) and histopathological analysis of liver ;
The content and activity of GSTA1 in serum and liver were evaluated by using serum transaminase ( ALT , AST ) , liver tissue index ( SOD , MDA , GSH , GSH - Px , TG ) and histopathological analysis .

The results show that :

1 . The acute liver injury model of CC14 - induced acute liver injury could be successfully replicated by 10 mL 路 kg - 1 dose of 0 . 35 % CCl 4 oil . Compared with the control group , the activity of serum transaminase increased significantly in the model group ( P0.01 ) . The changes of the indexes in the liver tissues were significantly higher than those in the control group ( P0.01 ) . The content and activity of GSTA1 in the liver were significantly increased ( P0.01 ) , while the content and activity of GSTA1 in the liver decreased significantly ( P0.01 ) .

2 . The acute liver injury model of mice was successfully replicated at 200 mg 路 kg -1 APAP . The serum transaminase activity in the model group was significantly higher than that in the control group ( P0.01 ) . The changes of the indexes in the liver tissues were significantly higher than those in the control group ( P0.01 ) . The content and activity of GSTA1 in the liver were significantly increased ( P0.01 ) , while the content and activity of GSTA1 in the liver decreased significantly ( P0.01 ) .

3 . The model of acute liver injury in mice was successfully reproduced at the dose of 14 mL 路 kg - 1 at 50 % ethanol . The serum transaminase activity in the model group was significantly higher than that in the control group ( P0.01 ) . The changes of the indexes in the liver tissues were significantly higher than those in the control group ( P0.01 ) . The content and activity of GSTA1 in the liver were significantly increased ( P0.01 ) , while the content and activity of GSTA1 in the liver decreased significantly ( P0.01 ) .

This study successfully replicated animal models of acute chemical , drug and alcoholic liver injury . In the three models of acute liver injury , the content and activity of GSTA1 increased significantly in serum ( P0.01 ) .
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332

【引证文献】