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目的 改良成人成骨细胞消化培养方法 ,以满足在细胞学水平进行骨代谢性疾病研究的需要。 方法 取无菌骨碎片 ,先用胰蛋白酶消化多次 ,以去除血细胞和成纤维细胞 ;骨片经短期培养后 ,再次使用胰蛋白酶消化 ,得到大量纯净成骨细胞。细胞长至汇合后生成黑色结节。分别采用NPP底物法、12 5I标记放射免疫 (RIA)法和Ⅰ、Ⅲ型胶原顺序沉淀抽提、SDS -PAGE还原电泳法测定细胞上清中成骨细胞主要特征性分泌物 :大量碱性磷酸酶 (AKP)、骨钙素 (OCN)和Ⅰ型胶原。 结果 黑色结节经四环素染色 ,荧光显微镜下观察为骨结节特征性的金黄色。成骨细胞上清中AKP分泌量为 (2 76± 0 5 6 )IU/ml、OCN分泌量为 (1 875± 0 5 49)ng/ml,明显高于成纤维细胞 (P <0 0 1)。可检测到Ⅰ型胶原而无Ⅲ型胶原。 结论 此改良方法可以在短期内得到大量性状稳定成骨细胞 ,同时避免成纤维细胞混入。
Objective To improve the method of adult osteoblast digestion and culture, in order to meet the needs of bone metabolism disease research at the cytology level. Methods Aseptic bone fragments were obtained and trypsinized several times to remove blood cells and fibroblasts. After short-term culture, the bone fragments were digested with trypsin to obtain a large number of pure osteoblasts. Cells grow to confluence to produce black nodules. NPP substrate method, 125I labeled radioimmunoassay (RIA) method and type I and III collagen sequences were respectively used for precipitation and extraction. The main characteristic secretions of osteoblasts in the cell supernatant were determined by SDS-PAGE: a large amount of alkaline Phosphatase (AKP), osteocalcin (OCN) and type I collagen. Results The black nodules were stained with tetracycline, and the golden yellow color of the nodules was observed under a fluorescence microscope. The amount of AKP secretion in osteoblast supernatant was (2 76 ± 0 56) IU / ml and that of OCN was (1875 ± 0 494) ng / ml, which was significantly higher than that of fibroblasts (P 0 01 ). Type I collagen can be detected without type III collagen. Conclusion This improved method can obtain a large number of traits in the short term to stabilize osteoblasts while avoiding the incorporation of fibroblasts.