体外酒精性脂肪肝模型的建立和评价

发布时间：2018-04-25 19:13

本文选题：肝细胞 + 凝胶包埋培养　；参考：《浙江大学》2010年硕士论文

【摘要】：随着人民生活水平的提高,过量饮酒引起的肝脏损伤日益增多,已经成为影响公众健康的问题。尽管对酒精性脂肪肝(简称酒精肝)的损伤机理,包括脂肪肝诱因和形成过程等,已有较为全面的认识,然而,目前尚无有效控制和治疗酒精肝的方法。其原因在于,缺少合适的酒精肝模型,因此难以筛选和找到有效的防治手段。目前酒精肝模型以动物为主,建立模型一般需要八周时间,高成本和低效率限制了其应用。而体外酒精肝模型则可以在短时间(1-2天)内建立,具有成本低、适合快速筛选治疗药物的优势。但是,传统的体外酒精肝模型采用单层贴壁肝细胞培养方式,并以高浓度的乙醇进行短时间刺激,该方法大大偏离了体内乙醇慢性肝损伤的情况,不能模拟体内酒精肝的发生过程。本文针对传统体外模型的缺点,采用能长期维持肝功能的凝胶包埋培养肝细胞,通过条件优化和体内外比较验证该模型的可靠性,并用于筛选抗酒精肝的中药有效成分,为寻找有效的酒精肝治疗药物奠定基础。论文分为三部分。第一部分为酒精肝模型的建立,考察了不同细胞培养方式、培养基和乙醇浓度对模型的影响,并采用针形电极在线检测了有无ParafilmM封口膜密封的培养皿中乙醇挥发速率差异。结果发现,肝细胞在Parafilm M封口膜密封的培养皿内以Williams' medium E培养基和凝胶包埋培养的方式并经过48h的200 mM乙醇作用,可以反映乙醇导致的体内肝细胞损伤。第二部分对该模型进行了详细评价,分析了包括脂质积累、过氧化压力和CYP 2E1诱导等各项指标。研究表明,经过乙醇处理后,体外模型的损伤指标呈现与体内酒精肝一致的变化趋势,说明该模型可以表征体内酒精肝的情况。然后,使用针形电极对肝灌流中的乙醇浓度变化(表征体内肝脏中乙醇浓度)进行了检测,初步尝试关联体内外模型的乙醇浓度,进一步验证了体外模型的可靠性。第三部分采用已建立的模型进行保肝药物筛选,找到了一种有效抗酒精肝的中药有效成分——纳米水飞蓟宾。总之,本文建立的体外酒精肝模型,可用于抗酒精肝药物筛选,尤其在利用我国丰富中药资源发掘有效保肝药物方面,具有广阔的应用前景。
[Abstract]:With the improvement of people's living standard, liver injury caused by excessive drinking has become a public health problem. Although there has been a comprehensive understanding of the mechanism of alcoholic fatty liver injury, including the inducement and formation process of fatty liver, there is no effective method to control and treat alcoholic liver. The reason is that it is difficult to screen and find effective prevention and treatment because of the lack of suitable alcoholic liver model. At present, the alcoholic liver model is mainly animal. It usually takes eight weeks to establish the model, and its application is limited by its high cost and low efficiency. The model of alcoholic liver in vitro can be established in a short time of 1-2 days. It has the advantages of low cost and suitable for rapid screening of drugs. However, the traditional model of alcoholic liver in vitro was cultured by monolayer adherent hepatocytes and stimulated with high concentration of ethanol for a short time. This method deviates greatly from the situation of chronic liver injury caused by ethanol in vivo. It is not possible to simulate the development of alcoholic liver in vivo. In view of the shortcomings of the traditional model in vitro, the hepatocytes were cultured with gel entrapment which could maintain liver function for a long time. The reliability of the model was verified by optimizing the conditions and comparing in vivo and in vitro, and it was used to screen the effective components of traditional Chinese medicine for anti-alcoholic liver. In order to find effective alcohol liver treatment drugs to lay the foundation. The paper is divided into three parts. The first part was the establishment of alcoholic liver model. The effects of different cell culture methods, culture medium and ethanol concentration on the model were investigated. The ethanol volatilization rate in the culture dish with or without ParafilmM sealing membrane was detected online by needle electrode. The results showed that the hepatocytes were cultured in Parafilm's medium E medium and gel in the culture dish sealed by Parafilm M sealing membrane and treated with 200mm ethanol for 48 h, which could reflect the damage of hepatocytes induced by ethanol in vivo. In the second part, the model was evaluated in detail, including lipid accumulation, peroxidation pressure and CYP 2E1 induction. The results showed that the damage index of the model showed the same change trend as that of the alcoholic liver in vivo after ethanol treatment, which indicated that the model could represent the alcoholic liver in vivo. Then, the change of ethanol concentration in liver perfusion was detected by needle electrode, which was used to characterize the concentration of ethanol in vivo and in vivo. The reliability of the model in vitro was further verified by correlating the ethanol concentration of the model in vivo and in vitro. In the third part, the established model was used to screen hepatoprotective drugs, and an effective Chinese medicine component, silibine-nanometer silybin, was found. In conclusion, the in vitro alcoholic liver model established in this paper can be used for screening anti-alcoholic liver drugs, especially in exploiting effective hepatoprotective drugs by using rich Chinese medicine resources in China, which has a broad application prospect.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R575.5;R-332

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