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目的:原代培养并鉴定大鼠骨髓间充质干细胞作为骨组织工程的种子细胞。方法:取大鼠骨髓,贴壁培养法培养。绘制生长曲线,检测细胞周期。real time PCR检测不同代数BMSCs胞内成骨相关因子mRNA水平。流式细胞仪检测细胞表面标志物。并行成脂及成骨诱导。结果:原代取材后24 h细胞即可贴壁,7 d可生长达培养皿底面积的80%。P3代BMSCs的生长曲线呈S形,近90%的细胞处于G0/G1期。从P0到P5BMSCs中几乎都有内源性bFGF、Shh、Cbfa1、ALP、OC的表达。流式细胞仪检测99.7%的BMSCs胞膜表面同时表达CD29及CD90,不表达CD45。并具有成骨分化及成脂分化的能力。结论:原代培养的BMSCs为骨组织工程提供了可靠的细胞来源。
OBJECTIVE: Primary culture and identification of rat bone marrow mesenchymal stem cells as seed cells for bone tissue engineering. Methods: Rat bone marrow and adherent culture were cultured. Draw a growth curve to detect cell cycle. Real time PCR was used to detect the mRNA level of intracellular osteogenesis related factors in different generations of BMSCs. Flow cytometry to detect cell surface markers. Parallel adipogenic and osteogenic induction. Results: The cells adhered to the surface 24 h after primary culture, and reached the basal level of 80% after 7 days. P3 generation of BMSCs growth curve was S-shaped, nearly 90% of cells in the G0 / G1 phase. Almost all P0, P5BMSCs have endogenous bFGF, Shh, Cbfa1, ALP, OC expression. Flow cytometry detected 99.7% of BMSCs membrane surface expression of CD29 and CD90, does not express CD45. And with osteogenic differentiation and adipogenic differentiation. CONCLUSION: Primary cultured BMSCs provide a reliable source of cells for bone tissue engineering.