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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10只,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 a method for isolation of rat Kupffer cells. Methods Ten healthy male Wistar rats aged 4 months and 200-250 g were used to isolate Wistar rat hepatocytes by modified Seglen collagenase perfusion method. Kupffer cells were isolated and purified by discontinuous density gradient centrifugation and selective adherent method. Inverted phase contrast microscope, transmission electron microscopy and fluorescence microscopy, and the application of phagocytosis and immunohistochemistry identification. Results The average cell yield of Kupffer cells was (3.37 ± 0.75) × 106 / g in liver tissue. The cell viability was (93.70 ± 1.49)% in Trypan blue staining. The newly isolated Kupffer cells were small round cells under optical microscope. Fluorescence microscopy showed no fluorescence under excitation light of 328 nm. Most of cultured 4 h shows that they have attached to the wall, increase the extension, appeared after 24 h pseudopodia, showed irregular shape. Kupffer cell nucleus under electron microscope kidney-shaped, cell membrane has many pseudopodia, phagocytosis, the cytoplasm contains larger secondary lysosomes. After 2 h of carbon ink engulfing test, the isolated cells were isolated from the tail vein of mice by intravenous injection of ink solution. The accumulation of black particles in the cytoplasm was found. ED2 expression was positive by immunohistochemical staining. Conclusion The rat liver Kupffer cells successfully isolated by the experiment are in good agreement with their morphological features, ultrastructure, phagocytic function and cell phenotype markers. The method is economical, efficient and stable.