兔骨髓间充质干细胞的分离、培养与鉴定

目的采用原代培养符合实验要求的兔骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）,进行生物学特性分析,为进一步骨组织工程实验研究奠定基础。方法采用全骨髓冲洗手段,结合密度梯度离心与贴壁培养方式体外分离兔BMSCs,倒置显微镜观察细胞形态,MTT绘制细胞生长曲线,ALP染色试剂盒检测细胞成骨性能。结果原代培养12d后可获得纯化的兔BMSCs,经成骨条件培养液诱导培养后,形态相对稳定,具有成骨性能。结论采用骨髓冲洗结合密度梯度离心与贴壁培养方式能够成功分离培养大量较纯化的兔BMSCs,第1～3代细胞体外培养生长状态好,形态稳定且贴壁率较高,可以作为后续骨组织工程实验种子细胞。

The isolation, culture and identification of mesenchymal stem cells from rabbit bone marrow

Objective To establish primary cells culture for rabbit bone marrow mesenchymal stem cells（BMSCs） to fit the requirement of experiments and analysis of BMSCs biological characteristics for further experimental study on bone tissue engineering.Methods Rabbit bone marrow mesenchymal stem cells were isolated by using whole bone marrow washing combining density gradient centrifugation and adherent cultivation methods.The morphology of BMSCs was observed in primary and passage cultures under inverted microscope;the growth curve of BMSCs was recorded by MTT;bone formation was identified by alkaline phosphatase staining kit.Results Purified BMSCs were obtained after 12 days of primary culture.The viable characteristics of BMSCs were relatively stable and the cells had bone formation potentials.Conclusion BMSCs can be isolated,purified and cultured from whole bone marrow by washing bone marrow cavity with density gradient centrifugation and adherent cultivation methods.The cultured cells grow stably during 1～3 passages with high rate attachment and good activity in vitro which can be used in the further experiments for bone tissue engineering study.