SD大鼠皮质骨来源间充质干细胞的培养与鉴定

Culture and Identification of SD Rat Cortical Bone Derived Mesenchymal Stem Cells

【目的】研究大鼠皮质骨来源间充质干细胞（MSCs）的分离培养及鉴定的方法。【方法】取SPF级体质量60～80 g的SD大鼠3只,运用骨髓贴壁法及胶原酶消化皮质骨法培养间充质干细胞,进行形态学观察、体外增殖能力考察、细胞周期分析、细胞表面抗原免疫荧光鉴定,并采用茜素红染色和油红O染色鉴定所培养细胞的成骨与成脂分化潜能。【结果】胶原酶消化皮质骨法及骨髓贴壁法均能有效分离纯化大鼠MSCs,经形态学、细胞表面抗原免疫荧光鉴定、成骨及成脂分化能力检测证实为间充质干细胞。【结论】自骨髓及皮质骨均能实现间充质干细胞的分离纯化,但皮质骨间充质干细胞来源广泛,原代不受血细胞干扰,是骨髓来源间充质干细胞的很好补充。

Objective To investigate the isolation and identification of SD rat cortical bone derived mesenchymal stem cells. Methods Mesenchymal stem cells（MSC） were isolated from six specific-pathogen free（SPF） rats weighing 60-80 g by collagenase digested method and by bone marrow adherent method separately. The morphology of bone marrow derived and cortical bone derived mesenchymal stem cells was observed, and the cell cycle was monitored for the analysis of their proliferation capacity in vitro. The cell surface markers were assessed by immunofluorescence, and the capacity of the cultured MSC differentiating into osteoblasts and adipocytes was investigated after alizarin red staining and oil red O staining. Results MSC could be effectively separated by the collagenase digested method and bone marrow adherent method. After morphological examination and identification by cell surface antigen immunofluorescence staining, the osteogenic and adipogenic cells were confirmed as MSC. Conclusion Both bone marrow and cortical bones can be used for the isolation of MSC. Rat cortical bone derived MSC have extensive sources, will not interfered by the blood cells in the stage of primary cells, and can be used as the supplement of bone marrow derived MSC.