人胚肋软骨静止区细胞的体外培养及生物学特性研究

Culture of the Resting Cell of Human Embryo Costal Cartilage and Study on Their Biological Behaviors in Vitro

目的 :研究体外培养的人胚肋软骨静止区细胞的生物学特性及分化规律 ,为组织工程生长板的研究提供种子细胞。方法 :采用Ⅱ型胶原酶消化的方法获取人胚肋软骨静止区细胞 ,观察细胞形态学变化 ;测定细胞生长曲线 ;借助特殊染色、免疫荧光染色的方法了解糖胺聚糖、碱性磷酸酶、Ⅱ型胶原酶的合成情况。结果 :贴壁后细胞呈多角形 ,从第 6代开始绝大多数细胞转变为成纤维细胞状 ,细胞培养至第 2代开始分泌大量Ⅱ型胶原。结论 :体外单层培养的人胚肋软骨静止区细胞具有正常的生长增殖与分化能力 ,可分化至成熟期软骨细胞。

Objective:To study the biological characteristics and differentiation rule of resting cell of human embryo costal cartilage cultured in vitro and provide the seed cells in large quantity for the use of human engineered growth plate tissue.Method:The embryo costal cartilage resting cells were harvested from 4-month-old human fetus by dissection and sequential digestion with 0.2% collagenase(type Ⅱ).The changes of cellular morphology were investigated.The proliferation of chondrocytes was observed by the growth curve.The biologic synthesis of the chondrocytes was determined by toluidine blue,alkaline phosphatase staining and type II collagen immunofluorescent reaction.The ultrastructure was observed by the electron microscope.The survival rate of the costal cartilage was observed after being frozen (-196℃).Results:After 24 hours the chondrocytes adhered to the bottles and the most cells became polygonal shape,a few appeared like a shuttle.Most of the cells transformed into the shape of the fibroblasts since the sixth generation,the cells of the second generation began secreting a great deal of the collagens (Ⅱ).Conclusion:The resting cells of human costal cartilage cultured in vitro can differentiate into the mature cells,the growth is normal and the cells can survive in the condition of deep-frozen.