小鼠Kupffer细胞分离及细胞培养

目的 采用在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离Kupffer细胞（Kupffer cells,KCs）,探讨其在分离小鼠KCs的应用及其对KCs生物活性的影响.方法 根据原位灌注和梯度离心方法不同随机分为4组：无胶原酶原位灌注＋3层梯度离心组（A）、无胶原酶原位灌注＋双层梯度离心组（B）、胶原酶原位灌注＋3层梯度离心组（C）和胶原酶原位灌注＋双层梯度离心组（D）.采用F4/80（BM8）免疫染色及吞墨实验判断细胞纯度和功能、台盼蓝拒染实验判断细胞的活力,探讨不同方法KCs分离的效果及细胞活性.结果 刚分离的KCs细胞近似圆形,接种l h后收获细胞纯度较高,但细胞得率相对较低.培养4 h后KCs得率相对较高,培养28 d仍能存活.免疫荧光可显示分离的为KCs,台盼蓝染色显示各组细胞的活力均在90 %左右,在体胶原酶灌注和双层梯度离心可以增加KCs的得率,双层梯度离心法可以增加分离KCs的纯度.结论 在体胶原酶灌注对提高KCs得率较为重要,在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离小鼠KCs的的方法简便、高效、稳定,培养的KCs具有良好的细胞生物学性状.

Isolation of Mouse Kupffer Cells and Their Primary Culture

Objective Kupffer cells （KCs） were isolated by perfusion of collagenase in vivo, discontinuous density gradient centrifugation, and selective adherence. The yield and purity of KCs were analyzed in different group to investigate the best isolated method of KCs. Methods Mouse KCs were isolated by collagenase digestion and the method were randomly divided into 4 groups according to the specific steps： without collagenase in situ perfusion and gradient centrifugation of three layer group （ A group） , double layer group （ B group） without collagenase in situ perfusion and gradient centrifugation of , collagenase perfusion in situ and gradient centrifugation of three layer group （ C group） and collagenase perfusion in situ and gradient centrifugation of double layer group （D group） . With F4/80 （BM8） and swallowing ink immunostaining experiments to deter- mine cell purity and function, using trypan blue dye test to determine cell viability, the survival time of KCs, morphological changes, as well as the effect of different methods were ob- served. Results KCs isolated freshly were just quasi-circular, and 1 h after inoculation cells were harvested at this time of high purity, but with relatively low cell yield. Four hours after inoculation KCs were harvested with relatively high yield. The cells were cultured for 28 d still alive. Trypan blue staining showed about 90% of cell vitality in each group. Immunohistochemistry and immunofluorescence showed that cells isolated were KCs. Conclusion Collagenase perfusion in vivo and gradient can increase the yield of KCs and gradient centrifugation of double layer solated KCs. In vivo perfusion of collagenase is important to improve the can increase the purity of iyield of KCs and separation of double layer can improve the isolated effect. The method for isolation and culture of Kupffer cells is convenient, efficient, stable and cultured Kupffer cells retain naive biological characteristics.