香豆素6胶束的制备及在单层上皮细胞摄取和跨膜转运研究

Preparation of Coumarin 6 Micelles and Uptake and Transport Study of Micelles Across Epithelial Cell Monolayer

目的探讨胶束在马丁达比犬肾(Madin Darby canine Kidney,MDCK)单层上皮细胞的摄取和跨膜转运机制。方法采用溶剂挥发法制备包载荧光探针香豆素6的聚乙二醇-聚乳酸(PEG3000-PLA3000)胶束,建立高效液相色谱法测定香豆素6含量的方法,考察空白胶束对香豆素6胶束的竞争摄取,考察温度、抑制剂对胶束在单层马丁达比犬肾细胞跨膜转运的影响,Ca2+浓度和乙二醇-双-(2-氨基乙醚)四乙酸(EGTA)的加入对胶束跨膜转运的影响。结果胶束可以很快被细胞摄取,胶束的细胞摄取可被空白胶束竞争抑制。胶束在单层细胞上的跨膜转运受温度和抑制剂的影响,说明胶束的跨膜转运是主动的、能量依赖性的过程;Ca2+浓度和乙二醇-双-(2-氨基乙醚)四乙酸可以影响胶束的跨膜转运,说明胶束可以经过细胞旁路途径跨膜转运。从单层细胞顶侧经跨膜转运到达基底侧的胶束的量相对于加入到顶侧的胶束量非常少。结论胶束在马丁达比犬肾单层细胞上的跨膜转运是细胞旁路途径和经细胞途径的结合。

OBJECTIVE To investigate the uptake and transport properties of micelles across Madin Darby canine kidney（ MDCK） cells. METHODS Coumarin 6 loaded PEG-PLA（ PEG3000-PLA3000） micelles were prepared by solvent evaporation method,and the HPLC determination method of coumarin 6 was constructed. Competition uptake of blank micelles with coumarin 6 micelles was studied. Temperature and inhibitors effect on transport of micelles across MDCK cell monolayer were studied,the concentration of Ca2 ＋ and EGTA were also studied. RESULTS Coumarin 6 micelles could be internalized into cells very quickly,and the uptake of micelles could be inhibited by blank micelles. The transport of micelles was affected by temperature and inhibitors,which means that this process is an active and energy-dependent process. The concentration of Ca2 ＋and EGTA could affect the transport of the micelles, which means micelles could transport across the cell monolayer via paracellular pathway. The amount of transported coumarin 6 micelles was rather limited compared with coumarin 6 micelles added to the apical side. CONCLUSION Micelles transport across MDCK cell monolayer via both paracellular and transcellular pathway at the same time.