鸡破骨细胞分离和培养方法的改进

Improvement of Culture Method for Chicken Osteoclasts

为获得大量有活力的鸡破骨细胞(Osteoclast,OC),本试验选取18日龄鸡胚,从长骨中提取骨髓细胞,用胰酶消化,40%和70%的percoll梯度离心分离骨髓间质细胞。在此基础上,用60 ng/mL核因子κB受体活化因子配体(RANKL)、50 ng/mL巨噬细胞集落刺激因子(M-CSF)诱导培养。采用抗酒石酸酸性磷酸酶(TRAP)染色、骨吸收陷窝以及标志性蛋白TRAP、基质金属蛋白酶9(MMP-9)、组织蛋白酶K检测鉴定破骨细胞。结果显示,诱导后的TRAP阳性细胞剧增,TRAP、MMP-9和组织蛋白酶K蛋白表达极显著上调,可在牛骨上形成大量的骨吸收陷窝。研究表明,该方法是一种快速、实用、高效的鸡破骨细胞分离培养技术。

To obtain viable chicken osteoclasts in large quantity for further in-depth study,bone marrow cells were isolated from tibia and femur of 18-day-age chicken embryos. The isolated cells were dispersed by trypsin digestion and centrifugated by percoll gradient centrifugation. Bone marrow stromal cells were collectedand cultured in α-MEM containing 60 ng/mL receptor activator of nuclear factor-κB ligand and 50 ng/mL macrophage colony-stimulating factor. Osteoclast formation was confirmed using tartrate-resistant acid phosphatase（TRAP）staining,bone lacunar resorption analysis and detection of the expression for TRAP,matrix metalloproteinase 9（MMP-9）and cathepsin K. The results showed that,a large number of TRAP-positive cells formed,the expression levels of TRAP,MMP-9 and cathepsin K sharply increased. Meanwhile,there were many resorption lacunae formed on the bovine bone. In conclusion,this method was a fast,practical and efficient way to obtain viable chicken osteoclasts in large quantity.