中国组织工程研究Issue(50)：8647-8653,7.DOI:10.3969/j.issn.2095-4344.2013.50.005
Ⅱ型胶原酶消化法培养兔关节软骨细胞
Culture of rabbit’s articular chondrocytes using type Ⅱ collagenase enzyme digestion method
摘要
Abstract
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. <br> OBJECTIVE:To investigate and improve the separation and culture method of articular chondrocytes of New Zealand rats at 4 weeks of age. <br> 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. <br> 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.关键词
组织构建/软骨组织构建/软骨细胞/体外培养/Ⅱ型胶原酶/骨关节炎/退变/表型/软骨/国家自然科学基金分类
医药卫生引用本文复制引用
闫虎,苏友新,林学义,陈宝军,周必洪,张庆..Ⅱ型胶原酶消化法培养兔关节软骨细胞[J].中国组织工程研究,2013,(50):8647-8653,7.基金项目
国家自然科学基金项目(面上项目)(81072828)@@@@the National Natural Science Foundation of China, No.81072828 （面上项目）
