改良后的大鼠原代肝细胞分离方法应用于格列喹酮肝摄取方式的考察

Study on hepatic uptake of gliquidone in primary rat hepatocytes by using improved isolation method

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 Seglen＇s method for isolating primary rat hepatocytes to develop a more convenient,efficient,economic and practical method,to investigate the uptake of gliquidone in primary rat hepatocytes.METHODS Under 37℃ and sterile conditions,primary rat hepatocytes were isolated by in situ and ex vivo infusion combined method,which was improved from Seglen＇s two-step in situ collagenaseⅣ perfusion method.Different concentrations of gilquidone were incubated with adhering primary rat hepatocytes for 40 min,which were then lysed with1% Triton X-100 for 1 h at 37℃.Then concentration of gliquidone in lysing solution was determined by HPLC-fluorescence,and amount of protein was determined by BCA method.Hepatic uptake curve of gliquidone was drawn to reveal uptake characteristics of gliquidone in primary rat hepatocytes.RESULTS The primary rat hepatocytes were efficiently and conveniently isolated by improved method,with regent use decreased,especially collagenaseⅣ.Cells harvested by this method had an activity 〉80% and benign morphologic characteristics of adhering hepatocytes.For gliquidone dose in the range of 15.625-720μg·ml^-1,hepatic uptake amount of gliquidone had linear relation with concentration of gliquidone.CONCLUSION The improved method can decrease use of regent,especially collagenaseⅣ,and it is more economic,effective and convenient.Hepatic uptake of gliquidone suggests that the main approach of gliquidone across hepatocellular membrane is free diffusion rather than tansporter mediated uptake.