【作者】 龚治；

【导师】 袁慧；

【作者基本信息】 湖南农业大学 ， 临床兽医， 2004， 硕士

【摘要】 由于对氨基苯胂酸广泛用于畜禽饲料添加剂中，不仅对畜禽产品造成残留，而且还对环境带来污染，日益威胁生态环境，为此我们进行了本研究。 研究目的：探讨有机砷剂——对氨基苯胂酸对体外培养的肝细胞的毒性损伤作用，并阐明其作用机理，推究对氨基苯肿酸诱导肝细胞凋亡的最低浓度，为对氨基苯胂酸的饲料添加标准提供更科学的理论依据，同时对其饲料卫生标准和食品安全性作出更准确的评价。 研究方法：本实验以大鼠原代肝细胞为实验模型，采用Seglen改良二步胶原酶灌注法分离肝细胞进行培养；用噻唑兰（MTT）法检测浓度分别为0、5、50、500μmol／L和5、10mmol／L的对氨基苯胂酸对大鼠原代肝细胞的杀伤作用；检测DNA Ladder梯状带以判定是否发生了凋亡；用黄嘌呤氧化酶法检测对氨基苯胂酸对肝细胞内和细胞培养液中的SOD活性的影响，同时分别测定细胞中GSH-Px活性和细胞培养液中H₂O₂的变化；用免疫组织化学法检测凋亡诱导蛋白Bax表达的改变。 实验结果：采用Seglen改良二步胶原酶灌注法分离肝细胞，其活率在85～95％之间；50μmol／L的对氨基苯胂酸对肝细胞的生长具有抑制作用，并有时间剂量依赖性；分别在24h、48h时检测到了DNA Ladder梯状带，说明对氨基苯胂酸诱导了肝细胞凋亡；细胞培养液中的SOD活性在50～500μmol／L的对氨基苯胂酸作用下明显降低，500μmol／L的对氨基苯胂酸能使细胞培养液中的H₂O₂活性在染毒后24h升高；5μmol／L和500μmol／L的对氨基苯胂酸分别使肝细胞内SOD和GSH-Px活性降低；免疫组化法显示，对氨基苯胂酸能使Bax的表达增高。 实验结论：对氨基苯胂酸能抑制体外培养的大鼠原代肝细胞的增殖，并能诱导肝细胞凋亡。对氨基苯胂酸诱发肝细胞凋亡的初步机理是抑制细胞抗氧化酶的活性，并促进凋亡蛋白Bax的表达升高。对氨基苯胂酸诱导肝细胞凋亡的最小浓度范围在50～500μmol／L之间。更多还原

【Abstract】 With the extensive use in addition agent of multipara, the arsanilic acid has resulted in remnant in products and environment pollution. So we carry out this research.Research Aim: dicuss toxicity trauma and function mechanism of arsanilic acid on cultured hepatocyte in vitro, and find minimum concentration range of arsanilic acid that can induce apoptosis of hepatocyte so that can provide theoretic basis on arsanilic acid’s additive standard and estimate of arsanilic acid’s feed sanitation and food safety.Research Methods: rat primary hepatocyte is used as empirical model. Rat hepatocytes were isolated by a single tow-step perfusion method. All the rat hepatocytes were treated with arsanilic acid at different mass concentrations (0, 5, 50, 500 u mol/L and 5, 10 mmol/L). Injury of hepatocytes was detected by MTT assay. Then apoptosis was observed by DNA agarose gel lelectrophoresls. The activity of SOD in hepatocytes and cell culture solution was determined by xanthine oxidase assay, and measured the git-up of GSH-Px and changes of H2O2 in cell culture solution. The changes of expression of Bax was determined by immunohistochemical method.Research Results: persistence of isolated hepatocytes was between 85% to 95%. The hepatocytes growing was inhibited by 50 U mol/L arsanilic acid in dose-dependent and time-dependent manners. And DNA Ladder was detected at 24h and 48h, that meaned the hepatocytes was induced by arsanilic acid. The git-up of SOD in cell culture solution was reduced by 50~500 u mol/L arsanilic acid. The cntent of H2O2 in culture solution was heightened by 500 U mol/L arsanilic acid after 24h. The git-up of SOD and GSH-Px in hepatocytes were reduced by 5 u mol/L and 500 u mol/L arsanilic acid respectively. The expression of Bax was boosted up by arsanilic acid.Research Conclusion: arsanilic acid effectively inhibits the proliferation of rat hepatocytes in primary culture and induced apoptosis. The restrain of SOD and GSH-Px and elevation of Bax expression were the mechanism of arsanilic acid inducing apoptosis. The minimum of concentration range of arsanilic acid inducing hepatocytes apoptosis was between 50 to 500 u mol/L.更多还原