转运葡萄糖苷类化合物的Caco-2细胞模型建立

Establishment of Caco-2 Cell Model for Transporting Glucoside Compounds

建立转运葡萄糖苷类化合物的Caco-2细胞模型,为葡萄糖及葡萄糖苷类化合物肠道吸收研究提供基础。通过对Caco-2细胞最适培养基的选择、单层致密性的测定、单层通透性和细胞两侧碱性磷酸酶活性比的测定、单层亚显微结构的观察、细胞葡萄糖转运蛋白SGLT1和GLUT2的mRNA及蛋白表达水平的分析,建立可靠的细胞模型。研究表明:Caco-2细胞的最适培养基为添加体积分数15%血清的DMEM低糖培养基;Caco-2细胞在14~17 d的跨膜电阻趋于稳定,苯酚红的表观渗透系数达到0.49×10^(-6)~0.51×10^(-6) cm·s^(-1),微绒毛分化成熟,细胞两侧碱性磷酸酶比值达到2.0以上。葡萄糖转运载体SGLT1的mRNA表达在14 d时显著高于17 d和21 d,葡萄糖转运载体SGLT1和GLUT2的蛋白表达在14、17、21 d无显著差异(P<0.05)。Caco-2细胞培养14~17 d即可建立可靠的肠细胞吸收模型,用于葡萄糖及葡萄糖苷类化合物吸收的体外研究。

Caco-2 cell model for the transport of glucoside compounds was established to provide the basis for studying intestinal absorption of glucose and glucoside compounds.A reliable cell model was established by selecting the best medium for Caco-2 cells,measuring the single layer density,detecting monolayer permeability and the activity ratio of alkaline phosphatase on both sides of the cell,observing the single layer sub microstructure,and analyzing the mRNA and protein expression levels of SGLT1 and GLUT2.The results showed that the optimal medium for Caco-2 cells was DMEM medium containing low glucose and supplying 15%serum.The trans epithelial electrical resistance of Caco-2 cells tended to be stable in 14-17 days,and the apparent permeability coefficient of phenol red reached 0.49×10^(-6)-0.51×10^(-6) cm·s^(-1).The microvilli differentiated and matured,and the ratio of alkaline phosphatase on both sides of cells reached more than 2.0.The mRNA expression of glucose transporter SGLT1 was significantly higher on 14 d than those on 17 d and 21 d,while the protein expression of glucose transporter SGLT1 and GLUT2 had no significant difference on 14 d,17 d and 21 d(P<0.05).Caco-2 cells could be cultured for 14-17 days to establish a reliable intestinal absorption model,which might be used to study the absorption of glucose and glucoside compounds in vitro.