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目的 :肝脏是肿瘤转移的好发部位 ,而肝脏含有丰富的免疫细胞 -枯否细胞 (Kupffer Cell,KC)。为研究枯否细胞的抗癌活性 ,旨在探讨 BAL B/ c鼠枯否细胞的分离培养方法。方法 :选用健康雄性 8～ 10周龄 BAL B/ c鼠 ,麻醉无菌取肝 ,注射冲洗去血并剪去部分结缔组织 ,小玻璃瓶中剪碎肝组织 ,加入 0 .0 4 %的 EDTA和 0 .0 5 %链霉蛋白酶 E溶液 ,交替轻轻吹打消化。不锈钢筛网滤过 ,用 0 .0 0 4 % Dnase 的 16 4 0液清洗细胞液 ,加 Tris-氯化胺溶解红细胞。 30 %～ 70 % Percoll梯度离心 ,10 % FBS贴壁培养 4～ 6 h洗去非贴壁细胞。通过对比体内、体外枯否细胞吞噬墨水和体外吞噬聚苯乙烯乳珠 (latexbeads,D1.1μm )鉴定枯否细胞。结果 :Kupffer细胞的得率为 (7.5 7± 1.92 )× 10 6 个 /鼠肝 ,即 6 .15× 10 6 个 / g,贴壁率为4 0 .18%。用 0 .4 %台盼蓝染色鉴定 ,细胞存活率在 95 %以上 ,吞噬鉴定发现 Kupffer细胞的纯度可达 90 %。贴壁 Kupffer细胞的形态多样 ,可见不规则 ,多边形 ,多角伪足 ,典型的星形及多角形。体外培养枯否细胞存活 2周后未见再有吞噬功能。结论 :酶消化法结合梯度离心和贴壁培养是分离 BAL B/ c鼠枯否细胞的可靠方法 ,为进一步实验打下了基础
Objective: The liver is a predilection for tumor metastasis, while the liver is rich in Kupffer Cell (KC) cells. In order to study the anticancer activity of Kupffer cells, we aimed to investigate the isolation and culture of BALB / c mouse Kupffer cells. Methods: BALB / c mice of 8 ~ 10 weeks old were selected from healthy males. Aseptic asepsis was used to remove the blood and the blood was removed by washing. Some of the connective tissue was cut off. The liver tissues were cut into small glass bottles and then added with 0.04% EDTA And 0 .0 5% pronase solution E, gently and gently digested. Stainless steel mesh filter, with 0. 0 0 4% Dnase 16 4 0 liquid washing cell solution, plus Tris-amine chloride dissolved red blood cells. 30% ~ 70% Percoll gradient centrifugation, 10% FBS adherent culture 4 ~ 6 h wash non-adherent cells. Kupffer cells were identified by comparing koilocytes in vitro and in vivo with swallowed polystyrene latex beads (latex beads, D1.1 μm). Results: The yield of Kupffer cells was (7.57 ± 1.92) × 10 6 cells / rat liver, which was 6.15 × 10 6 cells / g. The attachment rate was 40.18%. With 0 .4% trypan blue staining identification, the cell survival rate of 95% or more, phagocytic identification found that the purity of Kupffer cells up to 90%. Adherent Kupffer cells in various forms, showing irregular, polygonal, pseudopodia, typical star and polygon. Kupffer cells cultured in vitro after 2 weeks no further phagocytosis. CONCLUSIONS: Enzymatic digestion coupled with gradient centrifugation and adherent culture is a reliable method for isolating BALB / c murine Kupffer cells, laying the groundwork for further experimentation