摘要
目的研究猪肝细胞的获取率、活率．观测普通培养过程中肝细胞形态变化过程．方法取用本地杂种小猪作为肝细胞供体．用Ｓｅｇｌｅｎ胶原酶二步灌注法，原位灌注分离获取肝细胞悬液，ＴＢ染色法计算细胞数及细胞活率．在含１００ｍＬ／Ｌ胎牛血清及其他附助因子的ＤＭＥＭ培养基中培养，光镜下观察培养过程中肝细胞形态变化过程，检测不同时期培养上清中清蛋白、尿素的浓度．结果平均猪肝重４３４ｇ，每只猪肝平均可获取２１８×１０１０个肝细胞，平均活力为９４６％．在ＤＭＥＭ培养基中普通培养可存活４ｗｋ～５ｗｋ，并具有生物活性．结论本方法分离获取肝细胞产率高、活性高，可作为人工肝较好的生物材料．
AIM To study the yield and viability of porcine hepatocytes and observe the process of alteration of cell morphology in culture. METHODS Porcine hepatocytes were isolated by the modified two step method described by Seglen. The yield and viability were assessed by trypan blue exclusion. Hepatocytes were inoculated in the culture medium consisted of DMEM supplemented with insulin, glucagon, transferrin, dexamethasone and 100mL/L fetal calf serum. The morphologic change of cultured hepatocytes was observed and the concentrations of albumin and urea in the supernatant in different cultural period were examined. RESULTS The average liver weight was 434g , the average yield of porcine hepatocytes was 2 18×10 10 cells per pig with an average viability of 94 6%. Porcine hepatocytes were found to have differentiating function during in vitro culture for 4-5 weeks. CONCLUSION These data indicate that pig hepatocytes can be harvested with high yields and can retain viability and differentiating function for at least 30 days in vitro culture. Therefore it is an excellent source of hepatocytes for bioartificial liver devices.
出处
《世界华人消化杂志》
CAS
1999年第3期200-202,共3页
World Chinese Journal of Digestology
基金
国家自然科学基金
关键词
人工肝
肝细胞
细胞分离
细胞培养
artificial liver
hepatocytes
cell separation
cell, cultured
