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目的探讨从兔肋软骨膜和肋软骨中分别提取间充质干细胞的方法以及两者间的比较。方法取3～4周新西兰大白兔,共20只。酶消化及贴壁培养法分别获取软骨膜细胞和软骨细胞,体外培养观察其生长、形态、表型特征及其诱导成骨、成脂的分化潜能。结果从肋软骨膜中分离的原代细胞聚集成团,多为圆形和多边形;从第2代开始细胞以梭形为主,呈漩涡状排列,已传至第15代。从肋软骨中分离的原代细胞连接成片,以铺路石形态为主,第2代和第3代细胞逐渐转变成梭形;但第5代出现细胞老化,只传至第6代。免疫细胞化学染色发现,两种原代细胞均不表达CD29、CD90和CD34,从第2代开始均表达CD29、CD90,仍不表达CD34。原代软骨细胞表达Ⅱ型胶原,第2代细胞弱表达,第3代细胞开始不表达Ⅱ型胶原;而软骨膜细胞不表达Ⅱ型胶原。两种来源的第3代细胞经诱导均可分化为成骨细胞或者脂肪细胞。结论从兔肋软骨膜和软骨中均可获得干细胞。软骨细胞在体外可去分化成为干细胞。但软骨膜来源的干细胞的生长明显优于软骨来源的干细胞。
Objective To investigate the methods of extracting mesenchymal stem cells from rabbit costal cartilage and costal cartilage respectively and to compare the two. Methods Three to four weeks old New Zealand white rabbits were selected. The perichondrocytes and chondrocytes were obtained by enzymatic digestion and adherent culture respectively. The growth, morphology, phenotypic characteristics and differentiation potential of osteoblasts and adipocytes were observed in vitro. Results The primary cells isolated from the perichondrial periosteum aggregated into clusters with many circles and polygons. From the second generation, the cells were spindle-shaped and arranged in a spiral pattern and reached the 15th generation. The primary cells isolated from costal cartilage were connected into pieces, mainly in the form of paving stones. The second and third generation cells gradually transformed into spindle shape; however, the fifth generation appeared cell aging and passed only to the sixth generation. Immunocytochemical staining showed that neither of the two primary cells expressed CD29, CD90 and CD34, all expressed CD29 and CD90 from the second passage, and still did not express CD34. The primary chondrocytes expressed type II collagen, the second generation cells were weakly expressed, the third generation cells did not express type II collagen at first, while the perichondrocytes did not express type II collagen. Generation 3 cells of both origins can be induced to differentiate into osteoblasts or adipocytes. Conclusion Stem cells can be obtained from rabbit costal cartilage and cartilage. Chondrocytes can be dedifferentiated into stem cells in vitro. However, stem cells derived from perichondrocytes grew significantly better than those derived from cartilage.