成骨细胞超低温冻存前后生物学功能变化的研究

目的 观察超低温（液氮，-196 ℃）冻存对幼兔骨髓来源的成骨细胞体外培养的生长特性和生物学功能的影响。方法 自幼兔骨髓获取基质干细胞，体外诱导分化并培养 扩增，部分细胞进行液氮冻存，对冻存前后细胞的形态变化、增殖情况、克隆形成率、碱性磷酸酶（ALP）的合成能力、蛋白质合成能力及体外矿化能力进行对比观察，综合比较低温冻存对细胞功能的影响。结果 冻存复苏细胞的增殖有一定的时间延后性，其大体形态与冻存前基本一致，超微结构略有变化；复苏细胞仍保持典型的成熟成骨细胞的特征，具有较强的合成ALP及蛋白质的能力，两组细胞无统计学差异（P>0.05）；复苏细胞的体外矿化能力无明显变化。结论 超低温保存成骨细胞对其
生物学功能影响甚微，利用复苏细胞进行骨组织工程方面的研究是可行的。

Preliminary Study on Transformation of the Biological Function of the Cryopreserved Osteoblasts Cultured in vitro

Objective To culture and amplify the young rabbit′s bone marrow stromal cells（BMSCs） in vitro, and to observe the effect of hypothermia on the cells′ growing behavior and biological function. Methods BMSCs were acquired from the rabbit′ tibia bone marrow and induced to mature osteoblasts in vitro. The cultured cells growing well in vitro were preserved in liquid nitrogen. The anabiotic cells having cryopreserved for 1 week were chosen as the experimental group, and the routine 7th generation as the control group. Their biological function in comparion by the examination of morphological changes, cells′ proliferation ability, colone forming ratio, synthesis ability of ALP and protein, mineralized nodes forming ability were observed. Results As contrast to the control groups, the anabiotic cells also grew and proliferated well in vitro except a little more slowly than before. They had the similar general shape in all the time segments, but a little differences in cells′ ultrastructure. The experimental groups also had the typical characters of mature osteoblasts, and high abilities of the synthesis of ALP and proteins. The statistic data showed that these two groups had no significant difference（P>0.05）. Conclusion The cryopreserved osteoblasts had the same biological functions and the similar growing behaviors as before. Theseresults suggest that it is practical to use the cryopreserved osteoblasts for further study on bone tissue engineering.