小鼠肝枯否细胞的分离、培养与鉴定

The Seperation ,Cultivation and Identification of Mouse Kupffer Cell

目的研究BALB/c鼠肝枯否细胞分离、培养和鉴定。方法选用健康雌性8￣10w龄鼠,麻醉无菌取肝,D-Hank’s液注射冲洗去血并剪去部分结缔组织,剪碎肝组织,加入链霉蛋白酶E和I型胶原酶,水浴振荡消化,再加入DNaseI,共同消化。不锈钢筛网滤过,用0.005%DNaseⅠ的1640液清洗细胞液,加Tris-NH4CL溶解红细胞。30%￣70%Percoll梯度离心,用含10%FCS的RPMI-1640贴壁培养4￣6h洗去非贴壁细胞。通过吞噬墨水实验鉴定枯否细胞。结果枯否细胞的数量为(1.07±0.87)×107/g,贴壁率40.3%,用0.4%台盼蓝染色鉴定,细胞存活率在95%以上,吞噬实验发现90%的贴壁细胞具有吞噬功能,即KC的纯度可达90%。贴壁的枯否细胞的形态多样,已经完全伸出了伪足并且伸展贴壁,呈多边形、多角形或者梭形等不规则形状,并且胞浆内可见吞噬的微粒状物质。结论酶消化水浴振荡法结合梯度离心和贴壁培养是分离BALB/c鼠枯否细胞的可靠方法,为进一步实验打下了基础。

Objective To study the seperation,cultivation and identification of mouse Kupffer cell. Methods 8-10 weeks healthy female BALB/c mice were selected,and after anaesthesia we got the liver and took away the connective tissue and blood. The liver which was cut into pieces was mixed with Pronase E and Collagenase type Ⅰ. Then the mixture was shaken at 37℃ .After 20 minutes ,put DNase Ⅰ into the mixture. Filtration with stainless steel mesh was done before rinsing the cell and dissolving erythrocyte with Tris-ammoniachloride.Then gradient centrifugation were done with 30%-70% Percoll and cultivation with IO%FCS for 4-6 hours. The identification of Kupffer cell was contrasted with the phagocytosis of ink. Results The yield of Kupffer cell was （1.07 ± 0.87） × 10^7/g.Adhesive wall rate was 40.3%.Identification with 0.4% trypanblue showed that the cells survival rate was more than 95% and the purity was more than 90% in phagocytosis.The shape of Kupffer cell is of multiplicity, irregularity,polygon and multiangular. Conclusion Seperating Kupffer cell from BALB/c mouse by enzyme digestion combined with gradient centrifugation and adherent cultivation is a reliable methods ,and the work lays a foundation for study of liver metastasis.