【摘要】：目的:對(duì)分離大鼠原代肝細(xì)胞的經(jīng)典方法進(jìn)行改良,使之更方便、高效、經(jīng)濟(jì)和實(shí)用;并將方法改良后分離獲得的原代肝細(xì)胞進(jìn)行貼壁培養(yǎng)用于格列喹酮的肝細(xì)胞攝取方式的考察。方法:將雄性Wistar大鼠于無菌條件下麻醉后固定,保持體溫在37℃,對(duì)Seglen兩步在體灌注分離原代肝細(xì)胞方法進(jìn)行改進(jìn),采用先在體后離體的方法分離大鼠原代肝細(xì)胞,離心純化后進(jìn)行貼壁培養(yǎng);將不同濃度格列喹酮溶液與貼壁的原代肝細(xì)胞共同孵育40 min,1%Trion-X100裂解后,高效液相色譜-熒光法測(cè)定肝細(xì)胞裂解液中格列喹酮的濃度,BCA法測(cè)定細(xì)胞蛋白含量,繪制格列喹酮大鼠原代肝細(xì)胞攝取曲線,考察格列喹酮的肝細(xì)胞攝取方式。結(jié)果:經(jīng)改良后,所分離的大鼠原代肝細(xì)胞數(shù)量有所提高,活性80%,易于貼壁,形態(tài)良好,且節(jié)省試劑尤其是Ⅳ型膠原酶的用量;在15.625~720μg·ml-1范圍內(nèi)時(shí),隨著格列喹酮濃度的增大肝細(xì)胞對(duì)格列喹酮的攝取量呈近似線性的升高,并未呈現(xiàn)明顯的飽和現(xiàn)象。結(jié)論:經(jīng)典方法經(jīng)改良后,分離獲得的肝細(xì)胞活性高且易于貼壁,同時(shí)操作更為方便、高效,節(jié)約試劑尤其是昂貴的Ⅳ型膠原酶的用量,是較為經(jīng)濟(jì)、實(shí)用的方法;格列喹酮在15.625~720μg·ml-1范圍內(nèi)時(shí),肝細(xì)胞對(duì)其攝取以自由擴(kuò)散為主并非蛋白介導(dǎo)轉(zhuǎn)運(yùn)。
[Abstract]:Objective: to improve the classical method for the isolation of rat primary hepatocytes in order to make it more convenient, efficient, economical and practical. The primary hepatocytes isolated by modified method were used to observe the uptake of gliaquinone by adherent culture. Methods: male Wistar rats were anesthetized under aseptic conditions and maintained their body temperature at 37 鈩，

本文編號(hào)：2230922