三维组织化肝细胞模型反映非诺贝特的体内作用

发布时间：2018-01-03 03:03

本文关键词：三维组织化肝细胞模型反映非诺贝特的体内作用　出处：《浙江大学》2011年硕士论文　论文类型：学位论文

【摘要】：随着人们生活水平的提高,高血脂已经成为常见的现象。它是引起人类动脉粥样硬化等疾病的主要因素之一。因此,世界各国都很重视降脂药物的研发。肝脏是体内脂肪代谢的重要器官,也是降血脂药物调节脂肪代谢的主要作用位点和产生毒性的靶器官。在降血脂药物的研发过程中,药物对肝脏脂肪代谢的影响(即药效)和毒性是重点考察的内容。然而,体内实验成本高、周期长,因此需要有效的肝细胞体外培养模型,作为降脂药物药理和毒性评价的早期筛选工具。目前,已有的体外肝细胞模型包括三明治、聚球体和单层贴壁等。然而这些模型都有不可避免的缺陷,如三明治模型细胞间缺乏间隙连接,聚球体模型制备方法复杂、重复性差。而应用最广泛的单层贴壁模型,虽然操作方便且重复性好,但是由于缺乏与体内类似的微环境,其脂肪代谢水平与肝脏差异较大,且对药效和毒性也不够敏感。不仅如此,单层贴壁与三明治培养的肝细胞内脂肪代谢相关基因表达水平与体内肝细胞有较大区别。本文针对已有肝细胞体外模型的缺点,采用由本实验室自主研发,细胞间微环境更接近体内,并能长期维持细胞功能的三维凝胶包埋培养模型,进行肝细胞脂肪代谢以及典型降血脂药物-非诺贝特药理与毒理的体外研究,并通过与单层贴壁模型以及体内情况的对比验证该模型的可靠性。本文首先考察单层贴壁和凝胶包埋模型中肝细胞脂肪代谢的差别,发现凝胶包埋的肝细胞脂肪代谢更接近体内肝脏,证明了其作为脂肪代谢体外模型的可行性。其次,利用两种模型研究了非诺贝特的药理,结果显示非诺贝特对凝胶包埋培养肝细胞的脂肪代谢有明显的调节作用,但在单层贴壁培养模型无法体现。随后,本文评价了非诺贝特对两种模型肝细胞的短期毒性,结果发现凝胶包埋培养的肝细胞对非诺贝特的毒性更为敏感,且能正确反映体内毒性作用。而单层贴壁模型低估了非诺贝特应有的毒性。最后,采用能长期维持肝细胞功能的凝胶包埋模型进行了非诺贝特的长期毒性研究。结果显示,凝胶包埋模型能够反映非诺贝特的长期毒性和毒理,并同时显示了其毒理与其药理的关联性。总之,凝胶包埋模型能较好地模拟体内脂肪代谢,体现降血脂药物的药理,并能反映降血脂药物的短期和长期毒性。因此,该模型在降血脂药的筛选、安全评价和机理研究等方面有较好的应用前景。
[Abstract]:With the improvement of people's living standard, hyperlipidemia has become a common phenomenon. It is one of the main factors that cause human diseases such as atherosclerosis. Therefore, all countries in the world attach great importance to develop lipid-lowering drugs. The liver is an important organ of fat metabolism, is the main site of action of lipid lowering drugs regulate lipid metabolism and toxicity of the target organ. In the development process of lipid-lowering drugs, drug effects on liver lipid metabolism (i.e., efficacy and toxicity) is the focus of the content of the study. However, the in vivo experiment of high cost, long cycle, therefore need to be suitable in vitro models, as an early screening tool for the evaluation of the pharmacology and toxicity of lipid-lowering drugs. At present, in vitro models of hepatocytes include sandwich, spheroid and monolayer. However, these models have some unavoidable defects, such as lack of cellular sandwich model The gap junction, spheroid model preparation method is complex and poor repeatability. And the single most widely used wall model, although convenient operation and good reproducibility, but due to lack of microenvironment in vivo and similar, with the level of liver fat metabolism is different, and the efficacy and toxicity are not sensitive enough. Not only that, monolayer and sandwich cultured hepatocytes lipid metabolism related gene expression differences between hepatic cells in vivo.
Aiming at the disadvantages existing in vitro liver cell model, used by our laboratory, cell micro environment close to the body, the three-dimensional gel and can maintain cell function for long time embedding culture model in vitro for hepatic fat metabolism and typical hypolipidemic drugs - fenofibric special pharmacology and toxicology, and the reliability of the model the validation by comparison with the monolayer model and in vivo conditions.
This paper first studies the monolayer and gel entrapped hepatocytes fat metabolism model difference, found that hepatocyte lipid metabolism gel entrapped closer to the liver in vivo, proved its feasibility as fat metabolism in vitro model. Secondly, study the characteristics of fenofibric the pharmacology of two kinds of models, results show that the effects of fenofibrate on the gel pack the metabolism of fat liver cell culture embedded in a clear regulatory role, but in the monolayer culture model can not reflect the. Subsequently, the short-term toxicity of fenofibrate on two models of liver cells, the toxicity of gel entrapment culture of liver cells of fenofibrate is more sensitive, and can reflect the toxicity in vivo. The monolayer model underestimates the toxicity of fenofibrate should have. Finally, the embedding model of the long-term toxicity research of fenofibrate with liver cell function in long-lasting gel The results showed that the gel entrapment model could reflect the long-term toxicity and toxicology of fenofibrate, and showed the relationship between its toxicology and its pharmacology at the same time.
In conclusion, gel embedding model can simulate fat metabolism in vivo, reflect the pharmacology of hypolipidemic drugs, and reflect the short-term and long-term toxicity of hypolipidemic drugs. Therefore, this model has a good application prospect in screening, safety evaluation and mechanism research of hypolipidemic drugs.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R-332

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