基于Caco-2细胞的外源核酸吸收模型的建立及其吸收机制

基于人结肠癌细胞系Caco-2细胞建立外源核酸的吸收模型，并在此模型的基础上探究质粒DNA的肠道吸收机制。在Transwell孔膜上培养Caco-2细胞21 d后，通过透射电子显微镜观察细胞形态、监测培养期间跨膜电阻（transepithelial electrical resistance，TEER）值、测定荧光黄透过率评价细胞模型是否成功建成；通过细胞毒性实验探究质粒对细胞生长的影响；将质粒DNA加入细胞模型中进行双向转运实验，探究质粒的肠道吸收规律；在低温和添加特异性抑制剂条件下进行质粒转运实验，进一步推测质粒的肠道吸收机制。结果表明，细胞分化形态良好、TEER值及荧光黄表观透过系数（apparent permeability coefficient，Papp）均符合要求，细胞模型可用于转运实验；质粒对细胞生长无显著抑制作用，可用于转运实验；随着时间的延长，质粒在模型两方向上的转运均呈逐渐饱和趋势，Papp（肠腔侧（apical，AP）→基底侧（basolateral，BL））远大于Papp（BL→AP），提示质粒的转运受到某种载体蛋白的介导，膜介导的跨细胞运输方式可能参与其中；在低温和添加特异性抑制剂条件下，质粒的转运均受到显著抑制，进一步表明该过程是一个跨细胞方式的主动运输过程。

Absorption Model and Mechanism of Exogenous Nucleic Acids Based on Caco-2 Cells

In this study, we employed Caco-2 cells to establish an absorption model of exogenous nucleic acids. Using thismodel, we also explored the mechanism of intestinal absorption of plasmid DNA. After culturing Caco-2 cells for 21 dayson Transwell membranes, cell morphology, transepithelial electrical resistance (TEER) and apparent permeability coefficient(Papp) of fluorescent yellow were measured and used to evaluate whether the model was established successfully. Cytotoxicityexperiments were used to explore the effect of plasmid DNA on cell growth. The mechanism of intestinal DNA absorptionwas explored by two-way transfer experiments. To further validate vesicle-mediated DNA transport, transport experimentswere performed on ice or in the presence of the specific inhibitor. The results showed that the morphology of differentiatedcells was good and both TEER and Papp of fluorescent yellow reached the requirements; therefore the model could be appliedin transport experiments. The plasmid DNA showed no significant inhibitory effect on the growth of Caco-2 cells. As timepassed, the transport of plasmid DNA gradually tended to saturate in both directions. Papp (AP→BL) was much greater thanPapp (BL→AP), indicating that the transport was related to some carrier-mediated protein, and the transmembrane transportmode may be involved. These results further demonstrated that the transport of exogenous plasmid DNA was evidentlyinhibited by low temperature and the specific inhibitor and therefore a transcellular active transport process.