BACKGROUND:At present, the separation and culture technique of chondrocytes has been mature, but the chondrocytes grow slowly which are prone to degenerate using the present technique. It is not conducive to the fol ow-up test. OBJECTIVE:To investigate and improve the separation and culture method of articular chondrocytes of New Zealand rats at 4 weeks of age. METHODS:New Zealand rats aged 4 weeks were selected to take cartilage tissues from the bilateral knees that were resected under aseptic condition. Chondrocytes were isolated by type Ⅱ col agenase enzyme digestion and mechanical isolation method. The cells were cultured and passaged, and then identified by morphologic observation, toluidine blue staining and type Ⅱ col agen enzyme immunohistochemical methods. Growth curve was pictured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. RESULTS AND CONCLUSION:Inverted microscope observation showed that the primary cultured chondrocytes adhered at 6 hours after cultivation. The monolayer formation occurred at 72 hours after cultivation, and the cells were ready to be passaged at 96 hours after cultivation. In the fourth generation, some cells represented a spindle-like appearance. In the fifth generation, most cells turned into irregular shape appearance, and cellproliferation capacity diminished. Toluidine blue staining showed that the nuclei of cultured chondrocytes were blue and cytoplasm was pale blue. Immunofluorescent staining showed that cultured chondrocytes had a positive expression of col agen type Ⅱ and the color was tawny. Proliferative rate of chondrocytes in the first to third generations had no differences (P<0.05), while differences were found compared with the fourth generation in 4-7 days (P<0.05) and the fifth generation in 1-7 days (P<0.05). The results indicate that type Ⅱ col agenase enzyme digestion and mechanical isolation method is successful for isolating, cultivating New Zealand rat articular chondrocytes in vitro, and the first to third generations can be the best choice for the experiments of knee osteoarthritis.%背景：目前关节软骨细胞的分离培养技术已经比较成熟，但是研究中发现通过目前技术培养的软骨细胞生长周期慢，容易出现退变现象，不利于后续试验的进行。 目的：改进并探讨4周龄新西兰大白兔膝关节软骨细胞的分离与培养的方法。 方法：无菌条件下取4周龄新西兰大白兔双侧膝关节软骨，采用Ⅱ型胶原酶消化并机械吹打的方法，分离关节软骨细胞并进行原代、传代培养；采用形态学观察，甲苯胺蓝染色以及Ⅱ型胶原免疫组织化学方法对关节软骨细胞进行鉴定；MTT法检测关节软骨细胞增殖情况。 结果与结论：倒置显微镜下见膝关节软骨分离的原代软骨细胞6 h后开始贴壁，72 h可形成单层，96 h即可传代；前3代软骨细胞表型稳定，增殖力良好；第4，5代软骨细胞增殖能力减弱，绝大部分细胞变为长梭形和不规则形状。甲苯胺蓝染色显示培养的软骨细胞细胞质染成浅蓝色，细胞核染成深蓝色；免疫组织化学显示软骨细胞Ⅱ型胶原呈黄褐色阳性表达；MTT法检测表明前3代软骨细胞增殖差异无显著性意义(P>0.05)，且第1-3代与4代软骨细胞在培养第4-7天时，吸光度值差异有显著性意义(P<0.05)，与第5代软骨细胞在培养1-7天时，吸光度值差异有显著性意义(P<0.05)。提示采用Ⅱ型胶原酶消化并机械吹打的方法能够获得大量生长速度快且不宜退变的新西兰大白兔膝关节软骨细胞，且培养的前3代新西兰兔膝关节软骨细胞最为适宜。
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