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目的探索一种纯度高、数量大、操作简便的大鼠原代成骨细胞培养方法,为后续的实验研究奠定基础。方法 2009年3—9月在中国科学院金属研究所生物实验室,选择出生24h内的大鼠胎鼠4只,摘取颅骨,采用胰蛋白酶和Ⅰ型胶原酶交替消化分离成骨细胞并进行传代培养。相差显微镜下观察原代培养的成骨细胞形态;并采用碱性磷酸酶(ALP)染色、Ⅰ型胶原免疫荧光染色及茜素红染色对培养的细胞进行鉴定。结果该方法培养出的细胞具有典型的成骨细胞形态特征,ALP染色、Ⅰ型胶原免疫荧光染色及茜素红染色均表现为阳性。结论酶交替消化法原代培养胎鼠成骨细胞,获得的成骨细胞纯度高、数量大,可作为一种相对可靠、有效的原代成骨细胞培养方法。
Objective To explore a method of culturing rat primary osteoblast with high purity, large quantity and simple operation, which lays the foundation for further experimental research. Methods From March to September 2009, four rat fetuses born within 24 hours of gestation were selected from the Institute of Metals, Chinese Academy of Sciences. The skull was removed and osteoblasts were separated by trypsin and type Ⅰ collagenase digestion. to cultivate. The morphology of primary cultured osteoblasts was observed under a phase contrast microscope. The cultured cells were identified by alkaline phosphatase (ALP) staining, type I collagen immunofluorescence staining and alizarin red staining. Results The cells cultured by this method showed typical characteristics of osteoblasts. ALP staining, type Ⅰ collagen immunofluorescence staining and alizarin red staining showed positive results. Conclusion The primary culture of fetal rat osteoblasts by the alternate digestion of enzyme can obtain the high purity and large number of osteoblasts obtained, which can be used as a relatively reliable and effective primary osteoblast culture method.