应用联合酶建立BALB/c鼠枯否氏细胞体外分离培养方法

目的用作用功效不同的酶联合消化分离BALB/c鼠枯否氏细胞(KC),建立简便易行,产量、活性、纯度较稳定的KC分离培养方法。方法将0.1%型胶原酶、0.2%链霉蛋白酶、0.01%Dnase酶按1∶1∶1比例混合原位灌注和离体消化肝组织,经Percoll梯度离心,贴壁培养纯化细胞,应用荧光显微镜观察、免疫组织化学染色、吞噬功能实验进行鉴定。结果KC获得率为(2~3)×106/鼠肝,细胞存活率达96%,免疫组化和吞噬实验鉴定细胞纯度达92%。KC贴壁后形态大小不规则,呈多角形或星形,免疫组织化学染色显示溶菌酶阳性,胞浆内见吞噬的碳素墨汁颗粒。结论用作用功效不同的酶联合消化KC,经梯度离心和贴壁培养,可获得高产量、高活性、高纯度的BALB/c鼠KC,为进一步研究KC在肝脏疾病中的作用提供可靠的细胞来源。

Mixed Enzyme Applied to Develop the Method on BALB/c Mouse Kupffer Cell Isolated and Cultured in vitro

OBJECTIVE: To research the reliable method for the isolation and culture of Kupffer cell in BALB/c mouse by mixed enzyme. METHODS: Kupffer cells were isolated from liver by in situ perfusion and digestion with 0.1% IV collagenase, 0.2% pronase and 0.01% Dnase I, and by percoll density gradient centrifugation. Kupffer cells were identified by fluorescence microscope, immunohistochemistry and cell endocytosis effect. RESULTS: Kupffer cells were isolated successfully with high purity, the yield of (2-3) 10(6)/per mouse liver and the identification that 0.4% trypan blue indicated that the cells survival rate and purity were more than 96% and more than 92% respectively. The shape of Kupffer cell appeared to be multiplicity, irregularity, polygon and multiangular. Kupffer cells showed lysozyme positive by immunohistochemistry staining. And particles of India ink were found in cytoplasm. CONCLUSION: Here described technique for isolation and culture of Kupffer cells is simple and reliable, and can be used for preparing Kupffer cells with high yield, activity and purity.