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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。
Based on the model of human colon cancer cell line Caco-2, we established the absorption model of exogenous nucleic acid and explored the intestinal absorption mechanism of plasmid DNA based on this model. Caco-2 cells were cultured on Transwell membrane for 21 days. The morphology of the cells was observed by transmission electron microscopy. The transepithelial electrical resistance (TEER) value during the culture was monitored. The fluorescence yellow transmittance was evaluated to determine whether the cell model was successfully established. The effect of plasmid on cell growth was explored by cytotoxicity experiment. The plasmid DNA was added to the cell model for bi-directional transport experiments to explore the law of intestinal absorption of plasmids. Plasmid translocation experiments were carried out at low temperature and with specific inhibitors. Intestinal absorption mechanism. The results showed that the cell morphology was good, the TEER value and the apparent permeability coefficient (Papp) of TEER were satisfactory, and the cell model could be used for transport experiments. The plasmid had no inhibitory effect on the cell growth and could be used in the transport experiment. With the extension of time, the translocation of plasmids in both directions of the model tended to be saturated. Papp (apical, basal lateral, basal lateral, BL) was much larger than that of Papp (BL → AP) Was mediated by some kinds of carrier proteins, and membrane-mediated transcellular transport might be involved. Plasmid transport was significantly inhibited at low temperature and with specific inhibitors, further indicating that the process is a trans-cellular The way the active transport process.