肝细胞在纤维支架上的三维培养及评价

发布时间：2018-05-01 19:08

本文选题：原代肝细胞 + 三维培养　；参考：《西南交通大学》2014年硕士论文

【摘要】：药物研发过程中需要在早期利用体外肝模型考察其代谢和毒性情况,以确定是否进入后续研究。具有体内相关性的体外研究数据能够减少失败和消耗,提高研发效率和成功率。然而,目前体外原代肝细胞培养体系不能较好的保持肝细胞的极性和功能,特别是与药物代谢相关酶的活力。这样,由体外模型得到研究结果与体内数据存在较大差别,易引起误判。本研究旨在建立一种新型肝细胞体外培养模型,以较好地维持体外肝细胞的活性和功能,使体外模型研究结果能够更好地反映体内情况。电纺纤维支架能够较好的模拟细胞外基质的物理结构,是一种良好的细胞培养支架,但常规制备方法获得的支架孔径较小,不利于细胞渗透而并不能提供三维生长环境。本论文利用转筒收集方式制备大孔纤维膜,并改变转筒转速获得不同孔径的纤维支架。纤维表面经半乳糖、细胞粘附肽接枝改性,进一步模仿细胞外基质的生化环境特征。通过聚苯乙烯马来酸酐/聚苯乙烯混纺,改变纤维表面酸酐基团密度以获得具不同接枝密度的半乳糖改性纤维支架。通过改变细胞粘附肽反应加入量,制备具有不同RGD接枝密度的改性纤维支架。进一步通过先后两步反应制备具有不同比例的半乳糖和RGD共同接枝改性的纤维支架。肝细胞在支架上的生长情况表明,大孔纤维支架上肝细胞能够渗透进入支架内部,聚集成团,在三维空间上形成细胞-细胞、细胞-支架间的广泛的相互作用,肝细胞极性恢复。考察不同支架上培养15天内肝细胞物质合成和酶活力,发现孔隙率居中、RGD/半乳糖接枝比例为1：1000(密度分别约为7pmol/cm2和6nmol/cm2)的共接枝改性纤维支架上,肝细胞的功能保持最好。在有/无酶诱导剂或抑制剂情况下,研究酶代谢特异性药物睾酮、对乙酰氨基酚的体外代谢情况和胺碘酮、对乙酰氨基酚的肝细胞毒性反应,表明该培养模型能够较好地维持肝细胞酶活力、毒性敏感性和诱导/抑制响应性,具有一定的体内相关性,有望用于体外药物评价方面的研究。
[Abstract]:In the process of drug development, it is necessary to use an in vitro liver model to examine its metabolism and toxicity in order to determine whether to enter the follow-up study. In vitro research data with body correlation can reduce failure and consumption, improve the efficiency and success rate of research and development. However, the current primary hepatocyte culture system in vitro can not maintain liver cells better. The purpose of this study is to establish an in vitro culture model of a new type of hepatocyte in order to maintain the activity and function of the liver cells in vitro, and to make the results of the model in vitro model possible. Better reflect the situation in the body.
The electrospun fiber scaffold can well simulate the physical structure of the extracellular matrix, and it is a good cell culture scaffold. However, the diameter of the scaffold obtained by the conventional preparation method is small, which is not conducive to the infiltration of the cells and can not provide the three-dimensional growth environment. The fibrous scaffold with the same aperture. The fiber surface is modified by the graft of galactose and cell adhesion peptide to further imitate the biochemical characteristics of the extracellular matrix. By blending polystyrene maleic anhydride / polystyrene blend, the fiber surface anhydride group density is changed to obtain the different grafting density of galactose modified fiber scaffold. By changing the cell adhesion, the cell adhesion is changed. The modified fiber scaffolds with different RGD grafting densities were prepared with the addition of peptide reaction, and the fiber scaffolds with different proportions of galactose and RGD were prepared by two steps.
The growth of hepatocytes on the scaffold shows that the hepatocytes on the macroporous scaffold can penetrate into the stent and gather into groups to form cells and cells in the three-dimensional space, the extensive interaction between the cells and the scaffolds and the polarity recovery of the liver cells. The synthesis of liver cells and the activity of the enzymes in 15 days on the different scaffolds are investigated and the pores are found. RGD/ galactose graft ratio is 1:1000 (7pmol/cm2 and 6nmol/cm2) on the co graft modified fiber scaffolds. The function of hepatocyte remains the best. In the case of / without enzyme inducer or inhibitor, the enzyme metabolite drug testosterone, the metabolism of acetamiol in vitro and amiodarone, acetyl ammonia, The hepatocyte toxicity of base phenol shows that the culture model can maintain the enzyme activity, toxicity sensitivity and induced / inhibitory responsiveness of liver cells, and has certain in vivo correlation. It is expected to be used in the study of drug evaluation in vitro.

【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R318.08

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