Objective To investigate the experimental methods of isolation ,culture and identification of osteoblasts from neo-natal New Zealand rabbits in vitro .Methods Two-step enzymatic digestion was adopted to isolate osteoblasts from skull tissue of neonatal New Zealand rabbits to conduct primary cultured .Inverted phase contrast microscope was employed to study the cellular morphology ,acridine orange fluorescent staining was used to detect the cell adhesion function ,methyl thiazolyl tetrazolium(MTT) assay was employed to measure their proliferation ,and Alizarin red and tetracycline staining were used to test their mineralization . Results Primary osteoblasts were successfully obtained .Inverted phase contrast microscopy showed non-adherent cells were round ,while adherent cells were irregular fusiform ,triangular or polygonal .Acridine orange staining showed the nuclei of osteo-blasts green fluorescence ,with good adhesion ability .Good mineralization ability was also demonstrated by tetracycline and alizarin red staining .Osteoblasts possessed good proliferation activity .Conclusion Utilization of two-step enzymatic digestion contributes to getting a lot of osteoblasts with typical morphological features and biological activity in a short time .%目的：探讨新西兰乳兔成骨细胞体外分离、培养及鉴定的实验方法。方法采用二次酶消化法从新西兰乳兔颅骨组织块分离成骨细胞并进行原代培养，采用倒置相差显微镜进行形态学观察，吖啶橙荧光染色检测其黏附功能，采用四甲基偶氮唑盐（MTT）法检测其增殖情况，采用茜素红及四环素染色检测其矿化功能。结果成功获得原代成骨细胞；倒置相差显微镜显示未贴壁的细胞呈圆形，贴壁生长的细胞呈不规则梭形、三角形或多角形；吖啶橙染色可见成骨细胞的细胞核呈绿色荧光，具有良好的黏附能力；茜素红染色及四环素染色均显示其有良好的钙化能力；成骨细胞具有良好的增殖能力。结论采用二次酶消化法能在短时间内获得大量具有典型形态特征和生物学活性的成骨细胞。
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