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Caco-2细胞单层模型是研究药物吸收特性的重要工具,而P-糖蛋白(P-gp)对许多药物的药代动力学和药效学具有调控作用。对Caco-2细胞单层模型及P-gp功能进行研究,以便研究药物的体外转运机制。常规培养下,Caco-2细胞以2×105个/m L的密度接种400μL于Millicell培养皿中,通过观察细胞形态,绘制生长曲线,测定跨膜电阻值,检测细胞极性和罗丹明转运效率等对构建的细胞模型进行验证。结果:培养21 d后,细胞间形成紧密连接;跨膜电阻值达到恒定值,为(849±17)Ω·cm2;肠腔侧碱性磷酸酶活性显著高于基底侧酶活性,细胞形成极性分化,P-gp底物罗丹明在Caco-2单层细胞的表观通透系数(Papp)比值大于1.5(P(B→A)/P(A→B)>1.5),加入P-gp抑制剂后P(B→A)/P(A→B)小于1.5。研究表明,构建的Caco-2细胞模型结构和生化作用与小肠上皮细胞相似,细胞模型具有完整性、紧密性,细胞发生极性分化形成肠绒毛结构,且P-gp功能良好,可作为研究药物体外转运的模型。
The Caco-2 cell monolayer model is an important tool for studying drug absorption properties, whereas P-glycoprotein (P-gp) regulates the pharmacokinetics and pharmacodynamics of many drugs. Caco-2 cell monolayer model and P-gp function were studied in order to study the mechanism of drug in vitro transport. Under normal culture, Caco-2 cells were seeded in Millicell dish at a density of 2 × 10 5 cells / mL, and the growth curves were drawn. The transmembrane resistance was measured, and the cell polarity and rhodamine transport efficiency The constructed cell model was validated. RESULTS: Twenty-one days after culture, the cells formed tight junctions; the transmembrane resistance reached a constant value of (849 ± 17) Ω · cm 2; alkaline phosphatase activity in the intestine was significantly higher than that in basal side, The ratio of apparent permeability coefficient (Papp) of P-gp substrate rhodamine to Caco-2 monolayer was more than 1.5 (P (B → A) / P (A → B)> 1.5) P (B → A) / P (A → B) after gp inhibitor was less than 1.5. Studies have shown that the structure and biochemical effects of Caco-2 cells are similar to that of small intestine epithelial cells. The cell model has integrity and compactness, and cells differentiate to form intestinal villi. P-gp has good function and can be used as research drug In vitro transport model.