本文選題：原代肝細胞 + 三維培養(yǎng)��； 參考：《西南交通大學》2014年碩士論文

【摘要】：藥物研發(fā)過程中需要在早期利用體外肝模型考察其代謝和毒性情況,以確定是否進入后續(xù)研究。具有體內相關性的體外研究數(shù)據(jù)能夠減少失敗和消耗,提高研發(fā)效率和成功率。然而,目前體外原代肝細胞培養(yǎng)體系不能較好的保持肝細胞的極性和功能,特別是與藥物代謝相關酶的活力。這樣,由體外模型得到研究結果與體內數(shù)據(jù)存在較大差別,易引起誤判。本研究旨在建立一種新型肝細胞體外培養(yǎng)模型,以較好地維持體外肝細胞的活性和功能,使體外模型研究結果能夠更好地反映體內情況。 電紡纖維支架能夠較好的模擬細胞外基質的物理結構,是一種良好的細胞培養(yǎng)支架,但常規(guī)制備方法獲得的支架孔徑較小,不利于細胞滲透而并不能提供三維生長環(huán)境。本論文利用轉筒收集方式制備大孔纖維膜,并改變轉筒轉速獲得不同孔徑的纖維支架。纖維表面經半乳糖、細胞粘附肽接枝改性,進一步模仿細胞外基質的生化環(huán)境特征。通過聚苯乙烯馬來酸酐/聚苯乙烯混紡,改變纖維表面酸酐基團密度以獲得具不同接枝密度的半乳糖改性纖維支架。通過改變細胞粘附肽反應加入量,制備具有不同RGD接枝密度的改性纖維支架。進一步通過先后兩步反應制備具有不同比例的半乳糖和RGD共同接枝改性的纖維支架。 肝細胞在支架上的生長情況表明,大孔纖維支架上肝細胞能夠滲透進入支架內部,聚集成團,在三維空間上形成細胞-細胞、細胞-支架間的廣泛的相互作用,肝細胞極性恢復�？疾觳煌Ъ苌吓囵B(yǎng)15天內肝細胞物質合成和酶活力,發(fā)現(xiàn)孔隙率居中、RGD/半乳糖接枝比例為1：1000(密度分別約為7pmol/cm2和6nmol/cm2)的共接枝改性纖維支架上,肝細胞的功能保持最好。在有/無酶誘導劑或抑制劑情況下,研究酶代謝特異性藥物睪酮、對乙酰氨基酚的體外代謝情況和胺碘酮、對乙酰氨基酚的肝細胞毒性反應,表明該培養(yǎng)模型能夠較好地維持肝細胞酶活力、毒性敏感性和誘導/抑制響應性,具有一定的體內相關性,有望用于體外藥物評價方面的研究。
[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.

【學位授予單位】：西南交通大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R318.08

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本文編號：1830687