Colgalt2基因敲除小鼠Kupffer细胞形态及亚型差异

Differences of morphology and subtypes of Kupffer cells in Colgalt2 knockout mice

目的从Colgalt2基因敲除小鼠肝脏内分离Kupffer细胞,并比较其与正常小鼠Kupffer细胞形态分化和亚型分化的差异。方法采用胶原酶灌注法、percoll密度梯度离心法和免疫磁珠分选法分离纯化Kupffer细胞,并将分离的Kupffer细胞进行培养,显微镜下观察其形态的变化和差异。用脂多糖(lipopolysaccharide,LPS)分别刺激Kupffer细胞6小时、12小时和24小时,检测细胞亚型。结果分离获得Kupffer细胞数目为(4.42±0.63)×10~6/鼠肝,流式细胞仪检测细胞纯度为97.9%。培养后的细胞逐渐延伸为不规则形态,早期Colgalt2基因敲除小鼠Kupffer细胞形态分化相比正常小鼠有延迟,后期两者无显著差异。在未刺激状态下,Colgalt2^(-/-)小鼠M1型Kupffer细胞的比例小于Colgalt2^(+/+)小鼠,Colgalt2^(-/-)小鼠M2a型Kupffer细胞所占比例较高;LPS刺激6小时后,Colgalt2^(-/-)小鼠M2a型所占比例高于Colgalt2^(+/+)小鼠;LPS刺激12小时后,Colgalt2^(+/+)小鼠M2a型和M2c型Kupffer细胞所占比例小于Colgalt2^(-/-)小鼠;LPS刺激24小时后,Colgalt2^(-/-)小鼠M2c型细胞所占比例高于Colgalt2^(+/+)小鼠。结论应用胶原酶灌注法、percoll密度梯度离心法、免疫磁珠分选法可以获得纯度较高的小鼠肝脏Kupffer细胞,为后期的细胞实验奠定基础。Colgalt2基因的敲除会影响小鼠Kupffer细胞的形态分化和亚型分化。

Objective To compare the morphological differences between Kupffer cells which were isolated from Colgalt2 gene knockout mice and wild mice in primary culture. Methods Kupffer cells were isolated by perfusion of collagenase, density gradient centrifugation and immunomagnetic method. The morphology of the cells were observed by microscope. The subtypes of the Kupffer cells were detected at 6 h, 12 h and 24 h after incubated with lipopolysaccharide（LPS）. Results The average cell yield per mice liver was（4.42 ± 0.63） × 106. The purity of Kupffer cells was 97.9%. The cultured Kupffer cells exhibited irregular shape, in the early stages, the morphology of Kupffer cells isolated from Colgalt2 gene knockout mice exhibited later differentiation than wild mice, but in the later stages, it showed no difference. Under normal circumstances, the proportion of M2 a Kupffer cells in Colgalt2（-/-） mice were higher than that in Colgalt2（＋/＋） mice; the proportion of M2 a Kupffer cells in Colgalt2（-/-） mice were significantly higher at 6 hours after incubated with LPS; the proportion of M2a and M2c Kupffer cells in Colgalt2（＋/＋） mice decreased significantly than those in Colgalt2（-/-）mice after 12 houres. After 24 hours, the M2 c subtypes of Kupffer cells in Colgalt2（-/-） mice were significantly higher than those in Colgalt2（＋/＋） mice. Conclusions Higher purity of Kupffer cells were gained by perfusion of collagenase, density gradient centrifugation and immunomagnetic method. The Colgalt2 is involved in the morphology and the differentiation of Kupffer cells.