鼠肝Kupffer细胞的分离、培养和鉴定

Isolation, Culture and Identification of Kupffer Cells of Rat Liver

目的:Kupffer细胞是固定于肝脏的吞噬细胞,Kupffer细胞的分离、培养对肝脏疾病发生机制中的有关细胞和分子生物学的研究具有重要意义。方法:用链霉蛋白酶和胶原酶原位灌流,Nycodenz密度梯度离心分离大鼠Ku-pffer细胞,再经贴壁培养,并应用免疫组织化学、吞噬功能试验、电镜等方法进行鉴定。结果:本法能成功地获得高纯度的Kapffer细胞,Kapffer细胞得率为3～5×10~6/肝,贴壁后呈典型的星形及多角形,免疫组化染色示溶菌酶阳性、胞浆内见吞噬的印度墨汁及乳胶珠颗粒,电镜观察细胞表面有发达的伪足、微绒毛,胞浆内含大量溶酶体及吞噬的乳胶珠颗粒。结论:本实验所用的Kupffer细胞分离培养方法简单易行、可靠、细胞纯度高,可用于进一步研究Ku-pffer细胞的生物学功能。

Backgroung/Aims: Kupffer cells are hepatic macrophages that reside in the lumen of hepatic sinusoids. Isolation and culture of Kupffer cells are important for studing the pathogenesis of liver diseases. Methods: Kupffer cells were isolated from liver of Wistar rats by in situ perfusion with pronase and collagenase and density gradient centrifugation with Nycodenz, and maintenance cultures of purified Kupffer cells were established according to the attached time and cultured conditions; Kupffer cells were identificated by immunohistochemistry, endocytosis, and ultrastructure, etc. Results: Kupffer cells were isolated sucessfully with high purity, the yield was 3-5×106/liver. Kupffer cells showed particles of india ink and latex beads in cytoplasm, and lysozyme positive by immunohistochemistry staining. Kupffer cells had appearance with numerous lamellipodia, micro-filamentous and lysosomal structures, and latex particles of endocytosis under the transmission electron microscopy. Conclusions: The technique for isolation of Kupffer cells described here is simple, reliable, can be used for futher studing the biologic functions of Kupffer cells.