【作者】 吴月婷；

【导师】 孟琴；

【作者基本信息】 浙江大学 ， 生物化工， 2006， 硕士

【摘要】 本文以体外培养的原代肝细胞为模型研究药物肝毒性,并在此基础上进行保肝药物筛选。对比传统的肝细胞单层贴壁培养,本文采用了拟组织化的凝胶包埋培养方式,探究其在药物肝毒性和保肝药物研究中的应用可行性。本文分以下三部分: 1.采用凝胶包埋大鼠肝细胞研究5种不同机理的药物,即硫唑嘌呤、无味红霉素、红霉素、他克林和氯氮平的肝毒性作用,并与单层贴壁培养的结果比较。结果发现,1-5μM硫唑嘌呤、5-10μM无味红霉素、0.05-0.1mM他克林和36.7-73.6μM氯氮平均导致了凝胶包埋培养肝细胞的死亡,并伴随着肝细胞胞内谷胱甘肽(GSH)的显著降低。凝胶包埋培养的大鼠肝细胞对硫唑嘌呤的毒性反应不如单层贴壁培养敏感,而对无味红霉素和他克林的毒性比单层贴壁培养更敏感,推测这两种培养模型对不同毒性机理的药物毒性反应有明显差异。由于凝胶包埋培养中药物代谢酶活比单层贴壁培养高1.5-2倍,因此可能产生了更多的毒性代谢中间产物,所以凝胶包埋培养比单层贴壁培养对药物毒性敏感性高。而低浓度硫唑嘌呤在凝胶包埋体系中的传质可能是凝胶包埋大鼠肝细胞对硫唑嘌呤毒性不敏感的原因。5种药物的体外实验结果表明,凝胶包埋模型可以作为药物毒性研究的模型,并在药物毒性的中对药物毒性的机理有所提示。 2.以硫唑嘌呤损伤的鼠肝细胞作为肝毒性模型,研究凝胶包埋培养用于保肝药物筛选的可行性。GSH和N—乙酰半胱氨酸(NAC)对硫唑嘌呤作用48h的大鼠原代肝细胞没有保护作用,胞内GSH没有被增加。在两个模型中,5g/L甘草和0.15g/L甘草酸均能保持肝细胞内的GSH水平,减小硫唑嘌呤造成的肝细胞损伤,并且,将凝胶包埋中的肝细胞延长培养达96h,中药保肝药物的保护作用更加显著。因此,凝胶包埋培养模型可以用于保肝药物的筛选。 3.以人肝细胞模型研究硫唑嘌呤(1-50μM)体外肝毒性的结果与大鼠肝细更多还原

【Abstract】 This study, utilizing in vitro models, aimed to evaluate hepatotoxicity of several drugs in vitro and the effects of protective agents to suppress azathioprine hepatotoxicity. In vitro gel entrapment cultures of hepatocytes, which is one of the 3-dimentional and tissue-like cultures was employed to investigate its application in the aspect of drug safety/toxicity, and antihepatotoxic drugs evaluations. This study covers three aspects below:To investigate the applicablility of gel entrapment model in the drug safety/toxicity evaluations, five drugs was used which are azathioprine, erythromycin estolate, erythromycin, tacrine and clozapine and the two models, gel entrapment cultures and monolayer cultures, were compared. Results present that 1-5 μM azathioprine, 5-10 μM erythromycin estolate, 50-100 μM tacrine and 36.7-73.6 μM clozapine induce pronounced hepatotoxicity as well as severe cellular GSH depletion in the two in vitro model cultures of primary rat hepatocytes. Specifically, gel entrapment culture of primry rat hepatocytes seems to be less sensitive to azathioprine hepatotoxicity than monolayer cultures, while erythromycin estolate and tacrine injured gel entrapted hepatocytes much more severely than they did in monolayer culture. It seems like that drugs with different hepatotoxicity mechanisms perform different in the two in vitro culture models. Drugs which was induced by CYP450 shows more sensitive toxic effects to gel entrapment cultures because gel entrapment cultures maintains the more content of CYP450. Transport resistance of low concentration azathioprine in the gel entrapment culture could be one reason that lowered the toxic sensitivity of entrapted cells compared with the monolayer cultures We concluded with the five drugs results that the gel entrapment cultures of primary hepatocytes could be used in drug safety/toxicity evaluations.To investigate the antihepatotoxic drug effects in the azathioprine-induced hepatotoxicity, monolayer cultures and gel entrapment cultres were used and found that 5 g/Lliquorice and one of its bioconponents, 0.15 g/L glycyrrhizic acid, protect azathioprine-injured primary rat hepatocytes and inhibit cellular GSH depletion significantly. In prolonged gel entrapment cultures (96 h), liquorice and glycyrrhizic acid performed even well to maintain the cell viability and cellular GSH. Therefore, gel entrapment cultures could be used to evaluate the protective effects of the antihepatotoxic drug, especialy for the evaluations of Chinese medicines whose effects was very complicated or might be pronounced after long-term cultures.Results of primary human hepatocytes was very differents with rat hepatocytes in the test of 1-50 uM azathioprine hepatotoxicity utilizing monolayer cultures. Although cellular GSH was depleted profoundly in primary human hepatocytes, cell viability of 48 h treatment was not significantly decreased when azathioprine was lower than 10 uM. Considering the rare reports of severe toxicity of lower dose azathioprine in clinical therapy, it seems that primary rat hepatocytes may overestimated the risk of azathioprine.In conclusion, gel entrapment cultures of primary hepatocytes could be used prosperously in the field of drug safety/toxicity evaluations, protective drugs effect evaluations. Besides, drug toxicity could be different for human and experimental animals, primary human hepatocytes could be better than animal hepatocytes for the in vitro evaluation and investigation of drug hepatotoxicity.更多还原