背景：成骨细胞获取的方法较多，如何简便而迅速的获得高纯度的成骨细胞成为研究的热点。 目的：比较组织块法、胶原酶消化法、改良胶原酶和胰酶分段消化法体外培养纯化乳兔颅骨来源的成骨细胞结果及其细胞的生物学特点。 方法：取新生24 h内新西兰大白兔乳兔颅盖骨，采用组织块法、胶原酶消化法和改良胶原酶和胰酶分段消化法分离获取兔原代成骨细胞，并进行传代培养。通过倒置显微镜下形态学观察、锥虫蓝排斥法计数活细胞率及MTT法绘制细胞生长曲线、茜素红染色、细胞培养上清液碱性磷酸酶和骨钙素检测、Ⅰ型胶原和Ⅲ型胶原免疫组织化学法和RT-PCR检测骨钙素和Ⅰ型胶原mRNA的表达等对成骨细胞进行鉴定。 结果与结论：分离培养的成骨细胞均一性好、增殖能力强，具备成骨细胞的典型特征，茜素红染色阳性，Ⅰ型胶原免疫组织化学染色阳性，Ⅲ型胶原免疫组织化学染色阴性，细胞培养上清液碱性磷酸酶、骨钙素均有表达，PT-PCR结果有Ⅰ型胶原蛋白和骨钙素mRNA表达。改良胶原酶和胰酶分段消化法较传统胶原酶消化法有更高的细胞获取率，更好的细胞活性，且比传统胶原酶消化法耗时短(P<0.05)；组织块法操作方法最为简单，细胞活性最高，但是细胞产出率最低、耗时最长，不适合用于成骨细胞大规模培养。用改良的胶原酶和胰酶分段消化法可获得数量较多且纯度较高的成骨细胞，可以成为一种相对可靠、有效的原代成骨细胞的体外培养方法。%BACKGROUND:There are many kinds of ways to obtain osteoblasts at present, but how to get high-purity osteoblasts in a easy and fast way has become a hot research. OBJECTIVE:To explore a method to get massive and high purified osteoblasts effectively by comparing three common primary osteoblast culture methods, and to observe the biological characteristics of the osteoblasts from the skul of neonatal rabbit. METHODCalvarias were dissected from newborn New Zealand white rabbits within 24 hours, and osteoblasts were isolated with bone tissue method, col agenase digestion method and modified tryptase and col agenase sequential digestion method respectively, then the cells were subcultured in vitro. Osteoblast proliferation and osteogenic activity were identified by inverted microscope for morphology observation. The rate of living osteobalsts was counted with trypan blue staining. The growth curve of the cells was drawn with MTT method. Alizarin red staining was applied to detect alkaline phosphatase and osteocalcin protein in the cellculture supernatants. Col agen I and col agen III immunohistochemical staining was also performed. RT-PCR was used to determine the expression of osteocalcin and col agen I mRNA expression. RESULTS AND CONCLUSION:The cultured cells showed highly homogeneous appearance with active proliferation, and they had the typical features of osteoblasts. Alizarin red staining and col agen I immunohistochemical staining were both positive, while col agen III immunohistochemical staining was negative. Alkaline phosphatase and osteocalcin protein expression in the cellculture supernatants can be detected. The expression of osteocalcin and col agen I mRNA was positive in the RT-PCR test. Compared with col agenase digestion method, the modified tryptase and col agenase I sequential digestion method cost less time, presented higher production of osteoblasts and higher cellsurvival rate (P<0.05). Bone tissue method was the easiest method and did the least damage to osteoblasts, but it presented lowest production of osteoblasts and cost the maximum time among the three methods. So it cannot be used in large-scale osteoblast culture. A large quantity of high purity osteoblasts were obtained by modified trypsase and col agenase I sequential digestion method, which can be used as a reliable and efficient way to obtain the original generation osteoblasts in vitro.
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