色胺酮在Caco-2细胞模型上的吸收转运研究及Caco-2模型短期培养法的建立

发布时间：2018-05-27 06:29

本文选题：色胺酮 + Caco-2单细胞层　；参考：《复旦大学》2011年硕士论文

【摘要】：色胺酮属吲哚喹唑啉类生物碱,具有抗细菌、抗真菌、抗肿瘤等多种生物学活性,目前作为抗结核病及抗肿瘤候选药物已经进入临床前研究阶段。现有研究多集中在其药效及药理学研究,但是其药代动力学方面的研究几乎没有。本课题第一部分的研究内容是：使用体外培养的Caco-2单细胞层作为肠道吸收模型,研究色胺酮的肠道吸收转运特性。同时研究主要的外排蛋白P-糖蛋白(P-glycoprotein, encoded by MDR1, P-gp)和多药耐药相关蛋白2(multidrug resistance-associated protein 2, MRP2)是否参与色胺酮的肠道转运过程及色胺酮对P-gp及MRP2的表达及功能有何影响。结果显示,色胺酮在Caco-2模型上的吸收与外排转运都主要是通过被动扩散的方式进行,其从基底侧(BL, Basolateral)到肠腔侧(AP, Apical)以及从AP到BL的表观渗透系数Papp分别为4.731±0.101×10-5 cm/sec和6.138±0.291×10-5 cm/sec,外排系数Papp (BL→AP)/Papp (AP→BL)为0.77,表明其吸收方向(AP→BL)的转运占主导地位。P-gp抑制剂维拉帕米和MRP2抑制剂格列苯脲都没有对色胺酮的外排转运产生影响。而色胺酮在没有影响这两种外排蛋白的表达的情况下,抑制了P-gp底物地高辛及MRP2底物普伐他汀的外排转运。以上结果表明,色胺酮在Caco-2细胞模型上显示为由被动扩散介导的吸收良好的药物,主要的外排蛋白P-gp和MRP2都没有参与它的转运过程,而色胺酮对这两种外排转运蛋白的功能则具有一定的抑制作用。 Caco-2细胞模型是一种药物离体口服特性筛选模型,已广泛用于药物在小肠吸收的评价和各种转运机制研究中。该模型的建立一般要经过21天的培养周期,消耗大量时间和材料。而短链脂肪酸丁酸可促进Caco-2细胞的分化和细胞间紧密连接的形成。本课题第二部分的研究内容是：利用成分不同的含丁酸培养基建立一种短期培养Caco-2单细胞层的方法,并通过显微镜观察细胞形态学、测定跨细胞膜电阻和荧光黄通透量等指标对Caco-2单细胞层进行完整性评价,通过碱性磷酸酶活性检测对其进行细胞极化评价,通过RT-PCR方法评价对主要药物转运蛋白表达的影响。结果显示,使用短期培养法所得的Caco-2单细胞层形态完整,TEER500Ω·cm2,漏出标志物荧光黄Papp0.5×10-6 cm/s,表明单细胞层的完整性良好。同时AP侧碱性磷酸酶的活性显著升高,显示单细胞层出现明显的极性分化。短期培养法与21天培养法所得的Caco-2单细胞层中,药物转运蛋白P-gp和MRP2的RNA表达水平基本一致。表明了使用含丁酸的无血清培养基可促进Caco-2单细胞层的快速形成,该方法所建立的Caco-2细胞模型符合各项指标的要求,可用于研究口服药物的吸收、转运机制。
[Abstract]:Tryptophan is an indole quinazoline alkaloid with many biological activities, such as antibacterial, antifungal, antitumor and so on. Most of the current studies focus on pharmacodynamics and pharmacology, but there are few studies on pharmacokinetics. The first part of this thesis is to study the intestinal absorption and transport characteristics of tryptophan by using the single cell layer of Caco-2 in vitro as the intestinal absorption model. At the same time, whether the main efflux protein P-glycoprotein (encoded by MDR1, P-gp) and multidrug resistance-associated protein 2(multidrug resistance-associated protein 2 (MRP2) are involved in the intestinal transport of tryptophan and the effect of tryptamine on the expression and function of P-gp and MRP2 were studied. The results showed that the absorption and transport of tryptophan in Caco-2 model were mainly carried out by passive diffusion. The apparent osmotic coefficients (Papp) from basal BLL, Basolateralto AP, Apicaland from AP to BL were 4.731 卤0.101 脳 10-5 cm/sec and 6.138 卤0.291 脳 10-5 cm / sec.The efflux coefficient Papp BL / AP)/Papp / AP / L was 0.77, indicating that the transport of AP BLs in the absorption direction was dominant. P-gp inhibitor was dominant. Neither verapamil nor glibenclamide, an inhibitor of MRP2, had any effect on the transport of tryptamine. Tryptamine inhibited the efflux transport of P-gp substrate digoxin and MRP2 substrate pravastatin without affecting the expression of these two efflux proteins. These results suggest that tryptamine is a well absorbed drug mediated by passive diffusion in Caco-2 cell model. Neither P-gp nor MRP2 is involved in its transport process. Tryptophan has a certain inhibitory effect on the function of these two efflux transporters. Caco-2 cell model is a drug screening model in vitro, which has been widely used in the evaluation of drug absorption in the small intestine and in the study of various transport mechanisms. The establishment of the model usually takes 21 days of culture cycle and consumes a lot of time and materials. Short chain fatty acid butyric acid can promote the differentiation of Caco-2 cells and the formation of tight intercellular junctions. In the second part of this paper, the method of short-term culture of Caco-2 single cell layer was established by using different butyric acid medium, and cell morphology was observed by microscope. The integrity of Caco-2 single cell layer was evaluated by measuring the transmembrane resistance and fluorescence yellow permeability. The cell polarization was evaluated by alkaline phosphatase activity and the effect on the expression of major drug transporters was evaluated by RT-PCR method. The results showed that the morphology of Caco-2 single cell layer was intact and the fluorescent yellow Papp0.5 脳 10 ~ (-6) cm 路s ~ (-1), which indicated that the integrity of the single cell layer was good. At the same time, the activity of alkaline phosphatase in AP side was significantly increased, indicating obvious polar differentiation in single cell layer. The level of RNA expression of drug transporter P-gp and MRP2 in the single cell layer of Caco-2 obtained by short culture and 21 days culture was basically the same. The results showed that serum-free medium containing butyric acid could promote the rapid formation of Caco-2 single cell layer, and the Caco-2 cell model established by this method could be used to study the mechanism of absorption and transport of oral drugs.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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