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目的:对分离大鼠原代肝细胞的经典方法进行改良,使之更方便、高效、经济和实用;并将方法改良后分离获得的原代肝细胞进行贴壁培养用于格列喹酮的肝细胞摄取方式的考察。方法:将雄性Wistar大鼠于无菌条件下麻醉后固定,保持体温在37℃,对Seglen两步在体灌注分离原代肝细胞方法进行改进,采用先在体后离体的方法分离大鼠原代肝细胞,离心纯化后进行贴壁培养;将不同浓度格列喹酮溶液与贴壁的原代肝细胞共同孵育40 min,1%Trion-X100裂解后,高效液相色谱-荧光法测定肝细胞裂解液中格列喹酮的浓度,BCA法测定细胞蛋白含量,绘制格列喹酮大鼠原代肝细胞摄取曲线,考察格列喹酮的肝细胞摄取方式。结果:经改良后,所分离的大鼠原代肝细胞数量有所提高,活性>80%,易于贴壁,形态良好,且节省试剂尤其是Ⅳ型胶原酶的用量;在15.625~720μg·ml-1范围内时,随着格列喹酮浓度的增大肝细胞对格列喹酮的摄取量呈近似线性的升高,并未呈现明显的饱和现象。结论:经典方法经改良后,分离获得的肝细胞活性高且易于贴壁,同时操作更为方便、高效,节约试剂尤其是昂贵的Ⅳ型胶原酶的用量,是较为经济、实用的方法;格列喹酮在15.625~720μg·ml-1范围内时,肝细胞对其摄取以自由扩散为主并非蛋白介导转运。
OBJECTIVE: To improve the classical method of isolation of rat primary hepatocytes and to make it more convenient, efficient, economical and practical. Primary hepatocytes isolated after the improvement of methods were used for adherent culture for Gliquidone Investigation of liver cell uptake patterns. Methods: Male Wistar rats were anesthetized under aseptic conditions, the body temperature was maintained at 37 ℃, Seglen two-step in vivo perfusion method to improve the separation of primary hepatocytes, using the method of ex vivo isolated first rat Primary hepatocytes were isolated and purified by adherent culture. After incubated with different concentrations of gliquidone and primary hepatocytes adherent for 40 min, 1% Trion-X100 was lysed and analyzed by high performance liquid chromatography-fluorescence spectrometry Liver cell lysate gliquidone concentration, BCA determination of cellular protein content, draw Gliquidone rat primary liver cell uptake curve, to investigate the gliquidone liver cell uptake. Results: After the improvement, the number of isolated rat primary hepatocytes increased, the activity was> 80%, easily adherent, good shape, and save the reagent, especially the type Ⅳ collagenase dosage; in 15.625 ~ 720μg · ml -1, with the increase of gliquidone concentration, the uptake of gliquidone in liver cells increased approximately linearly without obvious saturation. CONCLUSION: The improved classical method is more economical and practical for isolated hepatocytes with high activity and easy adherence. At the same time, it is more convenient and efficient to operate, which saves reagents, especially expensive type IV collagenase. In the range of 15.625-720μg · ml-1, the hepatocytes up-regulated their free uptake and not protein-mediated transport.