作　　者： ; ; ; ;

机构地区： 南京军区南京总医院

出　　处： 《医学研究生学报》 2003年第11期822-824,I012,共4页

摘　　要： 目的 :分离及培养高纯度的原代大鼠肝细胞。　方法 :用含EGTA的D Hanks液及含DNaseⅠ的胶原酶消化液分两步原位灌注大鼠肝脏 ,分离细胞经 3次低速离心 (5 0 g ,5min)后获得纯化的肝细胞。肝细胞存活率检测用锥虫蓝拒染法 ,纯度检测用苏木精 伊红染色。采用加入特殊营养成分的RPMI 16 4 0培养液培养 ,用倒置相差显微镜观察细胞活力。　结果 :每只 180～ 2 0 0 g的大鼠平均可获 (1.0～ 1.5 )× 10 8的细胞 ,存活率 >5 0 % ,纯度高于98%。培养 1、3、5天肝细胞的存活率分别为 10 0 %、94 .5 %、89.5 % ,形态良好。　结论 :改良原位灌注消化法分离肝细胞产率较高 ,活性较好 ,加入特殊营养成分的RPMI 16 4 0培养液培养能保持肝细胞良好的形态。 Objectives: To establish a method of separation and culture of high-purity primary rat hepatocyte. Methods: The perfusion solutions used in the first and the second step were D-Hank's solution containing EGTA, and the digestive solution contained DNase I and collagenase respectively. Purified hepatocytes were separated from the dissociative cells by low-speed centrifugation (50 g, 5 min) 3 times. The survival rate was measured by typan blue exclusion and the purity was measured by routine hematoxylin and-eosin cytochemistry. Separated hepatocytes were cultured in RPMI 1640 culture medium with some special nutrient elements. Throughout the in vitro culture, we kept continuous observation of the shape changes of the hepatocyte and measured the survival rate within the first 5 days. Results:(1.0～1.5)×10 8 cells were prepared from a 180-200 g rat on average, the survival rate was higher than 50% and the hepatocyte purity was higher than 98%.Microscopically, the hepatocytes were normal in shape and viability. Conclusions: The modified in situ perfusion digestion method and the culture in RPMI 1640 and special nutrients are advantageous to get high viability and purity hepatocyte with normal shape.

关 键 词： 肝细胞 分离 培养 太鼠

领　　域： [生物学]