目的：改进与探讨新西兰大白兔软骨细胞体外培养的方法。方法：取3月龄新西兰大白兔自体血，制备自体血清备用，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应用新西兰大白兔自体血清进行原代、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膝关节软骨细胞进行鉴定，MTT法检测软骨细胞的增值能力；结果：倒置显微镜下见原代软骨细胞2 h后开始贴壁，8 h可形成单层，24 h即可传代。第1~5代细胞表型稳定，增殖力良好。甲苯胺蓝染色显示培养的软骨细胞核呈深染色，细胞质呈淡蓝色；免疫组织化学显示软骨细胞Ⅱ型胶原呈黄褐色表达，MTT检测显示前5代软骨细胞增值能力强，无明显差异；结论：结果表明自体血培养的前5代新西兰大白兔节软骨细胞均可用于膝骨关节炎的研究。%Objective:To investigate and improve the culture method of rabbit articular chondrocyte using autoserum in vitro.Methods: The 3-month New Zealand rabbit was killed to obtain the autoserum, 4-week New Zealand rabbit cartilage of bilateral knee was resected under aseptic condition. Chondrocyte was isolated by typeⅡcollagenase enzyme digestion and mechanical isolation method. The cell was cultured and passaged using autoserum, then identified by morphologic observation, toluidine blue staining, and typeⅡcollagen enzyme immunohistochemical, growth curve was pictured by MTT method. Results: It showed that the primary cultured chondrocyte adherented after 2 h under the Inverted microscope, and formed monolayer formation after 8 h. The chondrocyte was ready to be passaged after 24 h cultivation. The first to the fifth generation chondrocyte phenotype was stable, and the proliferation ability was well. Toluidine blue staining showed that the nucleus of cultured chondrocytes were blue and cytoplasm was pale blue; immunofluorescent staining showed that cultured chondrocytes had a positive expression of collagen typeⅡ that the color was tawny; proliferative rate of chondrocytes were detected by MTT showed that the OD value in the ifrst to iftfh generation had no differences (P<0.05).Conclusion:hTe research indicated that the ifrst to the iftfh generation chondrocyte was used to the research of knee osteoarthritis.
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