Analysis of Drug Permeation Mechanism across Oral Mucosa Using Cultured Stratified Cell Layers

为了阐明药物渗透口腔黏膜的机制，确定了HaCaT细胞分层培养的最佳条件，并对葡萄糖转运系统进行了各种基础研究，从增殖活性的角度选择了DMEM EGF(+)或DMEM+补充EGF(+)作为HaCaT细胞的培养条件。从组织学角度，选择DMEM EGF(+)。从跨粘膜电阻来看，DMEM：HAM的F-12 EGF(+)效果最好。综合考虑细胞存活率、层积、跨粘膜电阻和葡萄糖转运等因素，筛选出HaCaT细胞层的最佳培养条件为DMEM EGF(+)培养3~4周。细胞葡萄糖转运实验结果表明，细胞对D-葡萄糖的摄取速度明显快于L葡萄糖，表明葡萄糖转运蛋白在HaCaT细胞中的表达具有立体选择性。Western印迹分析表明，细胞内存在SGLT、GLUT1、GLUT2和GL3。然而，D-葡萄糖的跨细胞层转运速率与L-葡萄糖的跨细胞层转运速率没有显著差异。选择甘露醇、褪黑素和雌二醇作为被动转运药物，测定其PAPP值。三种药物的PAPP值与其亲脂性相关，提示培养的HaCaT细胞层可用于评价口腔粘膜对被动转运药物的通透性。

In order to clarify the drug permeation mechanism across oral mucosa, the optimal culture condition for the stratified cell layer using HaCaT cells was determined and the various basic studies were performed on glucose transport systems.As to the culture condition for HaCaT cells, DMEM EGF(+) or DMEM+Supplement EGF(+) was selected from the point of proliferative activity. From histological view point, DMEM EGF(+) was selected. From the transmucosal electrical resistance, DMEM : Ham's F-12 EGF(+) was the best. Taking factors such as cell viability, stratification, transmucosal electrical resistance and glucose transport into consideration, the best condition for HaCaT cell layers was selected to be the culture for 3 to 4 weeks using DMEM EGF(+).The result of glucose transport experiments using HaCaT cells showed that the uptake of D-glucose into cells was much faster than that of L-glucose, suggesting that the expression of stereo-selective glucose transporters in HaCaT cells. Western blot analysis showed that the presence of SGLT, GLUTs 1,2 and 3. Nevertheless, the transport rate of D-glucose across the cell layer was not significantly different from that of L-glucose. The reason for this observation is remaining unsolved.As the passively transported drugs, mannitol, melatonin and estradiol were selected, and the Papp values were measured. The results of Papp values for the three drugs were correlated with their lipophilicity, suggesting that the cultured HaCaT cell layers are effective for the evaluation of the oral-mucosal permeability to passively transported drugs.