大鼠肝Kupffer细胞分离方法改进  

Isolation method improvement of rat liver Kupffer cells

作　　者：张金卷[1] 朱争艳[1] 王毅军[1] 袁强[1] 孙伟[1] 沈云志[1] 李霖[1] 

机构地区：[1]天津市第三中心医院肝胆外科,天津市人工细胞重点实验室,天津300170

出　　处：《生物医学工程与临床》2015年第5期464-467,共4页Biomedical Engineering and Clinical Medicine

摘　　要：目的 建立大鼠Kupffer细胞分离方法。方法 选用4个月龄体质量200 ～ 250 g健康雄性Wistar大鼠，用改进的Seglen胶原酶灌注方法分离Wistar大鼠肝细胞，制成悬液。以Pecoll不连续密度梯度离心及选择性贴壁的方法分离纯化Kupffer细胞。用倒置相差显微镜、透射电子显微镜及荧光显微镜观察，并应用通过吞噬实验及免疫组织化学方法鉴定。结果 大鼠Kupffer细胞平均细胞产量为（3.37 ± 0.75） × 106/g 肝组织；Trypan blue染色检查细胞活性率为（93.70 ± 1.49） %。新分离的Kupffer细胞光学显微镜下呈小圆形细胞，荧光显微镜观察在328 nm波长的激发光下不发出荧光。培养4 h可见大部分已贴壁，增大延伸，培养24 h后出现伪足，呈不规则形。电子显微镜下Kupffer细胞细胞核呈肾形，细胞膜有许多伪足，吞噬活跃，细胞质中含有较大的次级溶酶体。碳素墨汁吞噬试验2 h后及大鼠尾静脉注射墨水溶液分离所得细胞，胞浆内均发现黑色颗粒聚积；免疫组织化学染色ED2表达阳性。结论 实验可成功分离的大鼠肝脏Kupffer细胞，符合其形态特点、超微结构、吞噬功能及细胞表型标志特点。方法经济、高效、稳定。Objective To establish the isolation method of rat liver Kupffer cells（KC）. Methods KCwere isolated from male Wistar rats（aged 4-month-old with body weight 200- 250 g） and purified by modified Seglen ＇s collagenase perfusion method,Pecoll gradient centrifugation and selective adherence of cells in culture protocol. The obtained cells were observed under inverted phase-contrast microscope and transmission electron microscope and identified by evaluation of phagocytes of ink phagocytes test and immune-histochemical staining. Results The mean cell yield was（3.37 ± 0.75） × 106/g liver,and viability was（93.70 ± 1.49） % by Trypan blue excluded test. Under light microscopy, the newly isolated cells showed round shape with small size.The cells were adherence in 4-hour of culture, and appeared to distend with irregular shape within 24-hour. There were numerous pseudopods in membrane of KC, kidney-like nucleus and cytoplasm contained many lysosomes under electron microscope. KC exhibited strong phagocyte activity and positive for ED2. Conclusion It is demonstrated that KCwith functional biological characters could be isolated and purified by introduced technique, and the cells meet the characteristic in morphology, ultra-microstructure, phagocytic function and cell phenotype. The introduced method is economic, efficient and stable.

关 键 词：肝脏非实质细胞 KUPFFER细胞 细胞分离 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]