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目的建立三维立体培养SD大鼠骨髓间充质干细胞(bone marrowmesenchymal stem cells,BMSCs)的方法。方法分离SD大鼠胫骨、股骨,冲洗骨髓腔,通过密度梯度离心并结合贴壁法培养BMSCs,流式细胞仪检测其生长周期,电子显微镜观察其内部结构,免疫细胞化学法进行细胞表面抗原标志物鉴定。后将其接种到细胞外基质(extracellularmatrix,ECM)胶中,加入DMEM-F12细胞培养液进行三维立体培养。结果培养的细胞呈梭形,成纤维细胞样,单核,增殖能力强。流式细胞术显示93.10%细胞处于G0～G1期。透射电镜结果显示细胞核质比例较大,核仁大而明显,细胞表面有较多微绒毛,细胞质细胞器丰富,表现出未分化细胞的特征。免疫组织化学结果显示细胞表达波形蛋白(vimentin)、纤维连接蛋白(fibronectin),不表达角蛋白(keratin)。BMSCs在ECM胶中生长良好。结论分离、培养的细胞为骨髓BMSCs,在三维培养体系中生长良好。
Objective To establish a method of three-dimensional culture of bone marrow mesenchymal stem cells (BMSCs) from SD rats. Methods BMSCs were isolated from the tibia and femur of the SD rats, and the bone marrow cavity was washed. The BMSCs were cultured by density gradient centrifugation and adherence method. The growth cycle was detected by flow cytometry. The internal structure was observed by electron microscopy and the cell surface antigen Material identification. The cells were inoculated into extracellular matrix (ECM) gel and cultured in DMEM-F12 cell culture medium for three-dimensional culture. Results The cultured cells were spindle-shaped, fibroblast-like, mononuclear and proliferative. Flow cytometry showed that 93.10% of cells were in G0 ~ G1 phase. Transmission electron microscopy showed a large proportion of nuclear cytoplasm, large nucleoli and obvious, more microvilli on the cell surface, abundant cytoplasmic organelles, showing the characteristics of undifferentiated cells. Immunohistochemistry results showed that the cells expressed vimentin, fibronectin and did not express keratin. BMSCs grow well in ECM glue. Conclusion The isolated and cultured cells are bone marrow BMSCs and grow well in the three-dimensional culture system.