基础研究大鼠原代肝细胞、星状细胞、枯否细胞和.PDF

·532 · doi 10.3969/j.issn. 1673-4254.2014.04.20 J South Med Univ, 2014, 34(4): 532-537 基础研究 大大鼠原代肝细胞鼠原代肝细胞、、星状细胞星状细胞、、枯否细胞和肝窦内皮细胞的同步分枯否细胞和肝窦内皮细胞的同步分 离与离与培养培养 李 洋，蔡双明，张莉莉，李 旭 南方医科大学南方医院急诊科，广东 广州 510515 摘要：目的探索和建立同步分离大鼠原代肝细胞、星状细胞、枯否细胞和肝窦内皮细胞，并进一步鉴定和培养的方法。方法 联 合应用原位灌注、离体灌注、梯度密度离心和差速贴壁等分离方法，获取4 种大鼠肝内原代细胞，采用形态学观察、免疫荧光染色 和墨汁吞噬实验等方法对所分离的各种原代细胞进行纯度鉴定。结果 通过原位灌注结合离体灌注、密度梯度离心结合差速贴 壁等分离手段，成功实现了大鼠原代肝细胞、星状细胞、枯否细胞以及肝窦内皮细胞的同步分离，且所得原代细胞得率、活力及 纯度等指标均符合后续细胞实验要求。结论 通过简易方法同步分离大鼠原代肝细胞、星状细胞、枯否细胞以及肝窦内皮细胞 是可行的。 关键词：原代培养；肝细胞；星状细胞；枯否细胞；肝窦内皮细胞；原位灌注；密度梯度离心 Simultaneous isolation and primary culture of rat hepatocytes, hepatic stellate cells, Kupffers cells and hepatic sinus endothelial cells LI Yang, CAI Shuangming, ZHANG Lili, LI Xu Department of Emergency Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China Abstract: Objective To establish a method for simultaneous isolation and primary culture of rat hepatocytes, hepatic stellate cells, Kupffers cells and hepatic sinus endothelial cells. Methods By combining in situ perfusion, in vitro perfusion, density gradient centrifugation and differential adhension, primary rat hepatocytes, hepatic stellate cells, Kupffers cells and hepatic sinus endothelial cells were obtained. The purity of these cells were assessed with morphological observation, immunofluorescent staining and ink phagocytosis assay. Results We successfully obtained the 4 primary cells simultaneously by combining in situ perfusion with in vitro perfusion, density gradient centrifugation, and differential attachment. The cell yield rate, cell viability and purity all met requirements for the subsequent cell experiment. Conclusion The combined cell isolation and culture method is feasible to isolate primary rat hepatocytes, hepatic stellate cells, Kupffers cells and hepatic