大鼠成骨细胞的原代培养和鉴定

Primary culture and identification of rat osteoblasts

背景:组织工程需要大量的种子细胞,成骨细胞已成为骨组织工程构建的重要种子细胞之一。但成骨细胞取材困难,获得的成骨细胞的纯度不一。目的:建立大鼠新生乳鼠颅骨来源成骨细胞的分离培养纯化方法,观察颅骨来源成骨细胞生物学特点。方法:采用二次酶消化法对SD大鼠乳鼠成骨细胞进行原代培养,扩增。通过差速贴壁法进行成骨细胞纯化。通过形态学、细胞碱性磷酸酶检测、茜素红染色、钙结节Vonkossa法染色、超微结构以及细胞增殖曲线,确定其增殖与成骨活性。结果与结论:二次酶消化法培养颅骨来源成骨细胞可获得原代细胞增殖,传代扩增细胞具有典型成骨细胞形态学和生物学活性。碱性磷酸酶、茜素红染色、钙结节Vonkossa法染色均呈阳性结果。超微结构显示为高分化功能活跃成骨细胞,细胞增殖曲线显示细胞生长活跃。提示,新生SD大鼠颅骨来源成骨细胞具有良好的增殖与成骨活性,能连续传代增殖,纯度高,细胞生物学特征稳定,适用于做体外实验研究。

BACKGROUND：Tissue engineering requires a lot of seed cells.Osteoblasts have become an important seed cells in bone tissue engineering.However,there are the difficulties to derive osteoblasts and the different purity of osteoblasts was obtained.OBJECTIVE：To establish the methods of isolated culture and purification of neonatal rat calvarial osteoblasts,and to observe the biological characteristics of the osteoblasts from the skull.METHODS：Osteoblasts of Sprague-Dawley neonatal rats were primarily cultured and proliferated by the second enzyme digestion.Osteoblasts were purified by differential attachment method.Osteoblast proliferation and osteogenic activity were identified by morphology,alkaline phosphatase detection,Alizarin red staining and calcium nodules Von kossa staining,ultrastructure and cell proliferation curve.RESULTS AND CONCLUSION：Primarily cultured calvarial osteoblasts could proliferate by the secondary source of enzyme digestion.The amplification cells showed representative morphological and biological characteristics of osteoblasts.Alkaline phosphatase,Alizarin red staining and calcium nodules Von kossa staining presented positive results.Ultrastructural features appeared as high-differentiated active osteoblasts.Cell proliferation curves showed active cells.Results indicated that calvarial osteoblast of Sprague-Dawley rat has good proliferation and osteogenic activity and can be continuously passaged,with high purity,cell biological characteristics of stability.Calvarial osteoblast is suitable for experiment in vitro.