自骨密质中分离、培养大鼠骨髓间充质干细胞

Isolation and Culture-Expansion of Rat Compact Bone-Derived Mesenchymal Stem Cells

目的介绍一种自Wistar大鼠骨密质中分离培养间充质干细胞(Mesenchymal stem cells,MSC)的方法。方法应用骨髓密度梯度离心、酶消化骨密质两种方法分离、培养Wistar大鼠MSC,比较其形态学、体外增殖能力差异;并用碱性磷酸酶染色和油红O染色分别鉴定MSC的成骨和成脂分化潜能。结果采用密度梯度离心骨髓或用酶消化后的骨密质,经贴壁培养后均能够成功分离和培养大鼠的MSC,两者在形态学和体外增殖能力方面无明显差异;且两种方法培养的MSC经特异性诱导剂诱导后,均可向脂肪及成骨细胞分化,油红O染色及碱性磷酸酶染色均阳性。结论自骨髓和骨密质中均能够分离培养出较为纯化的大鼠MSC,两种方法培养的MSC体外生物学性能无明显差异。

Objective To introduce a detailed protocol for isolating and expanding mesenchymal stem cells （MSC） from Wistar rat compact bones. Methods Wistar＇s MSC were isolated and culture-expanded from bone marrow by Percoll density gradient centrifugation or from compact bone debris by digestion with collagenase type II. The morphological and proliferation features between the two populations of MSC were compared. The in vitro osteogenesis and adipogenesis were identified by intracellular alkaline phosphatase activity and droplets of lipids respectively. Results MSC were successfully isolated and culture-expanded by two different methods. Both of these two populations took fibroblast-like morphology and exhibited similar proliferative activity. They could be induced into adipocytes and osteoblasts in vitro under the defined conditions, which were revealed by positive Oil-Red O and alkaline phosphatase staining. Conclusion Both bone marrow and compact bones could be served as the sources for isolating rat MSC, and the biological properties of the harvested cells are comparable.