新生小鼠颅盖骨间充质干细胞原代培养模型的建立

目的:探索新生小鼠颅盖骨间充质干细胞原代培养的方法?方法:取新生小鼠颅盖骨,采用胶原酶消化获得间充质干细胞,观察细胞的形态及增殖情况,通过细胞周期分析细胞增殖能力,通过流式测定细胞表面标志来分析细胞纯度,对细胞进行成骨成脂诱导分化,并分别予茜素红及油红O染色定性,荧光定量PCR检测成骨标志基因Osteocalcin及成脂标志基因PPARγ?Fabp4表达情况?结果:培养出的纺锤形细胞均一性好?增殖能力强?流式测定细胞表面标志显示细胞纯度好,高表达CD29?CD44,几乎不表达CD34?CD45?诱导分化后分化率高,茜素红染色及油红O染色分别可见较多矿化结节及脂滴,荧光定量PCR显示随分化相关标志基因均明显增高?结论:本研究成功建立了新生小鼠颅盖骨间充质干细胞原代培养模型,为进一步研究间充质干细胞奠定基础?

Establishment of culture model for primary mesenchymal stem cells from neonatal mouse calvaria

Objective:To establish a culture method for primary mesenchymal stem cells from neonatal mouse calvaria. Methods:Mesenchymal stem cells were collected from neonatal mouse calvaria with collagenase. The morphological changes and proliferation of the cultured cells were observed. Cell circle stages were analyzed by flow cytometry. The expression of cell-surface antigens were detected by flow cytometry. The potential of the isolated cells to differentiate into osteogenic and adipogenic lineages was examined. The mineralization and the intracytoplasmic lipid were detected using alizarin red staining and oil red O staining,respectively. Differentiation abilities were further validated by RT-PCR analysis of osteoblast specific gene and adipocyte specific genes. Results:The cultured spindle-like cells showed highly homogeneous appearance with active proliferation. The cells were positive for CD29 and CD44, while negative for CD34 and CD45. Visible mineralized nodules and lipid droplets were observed with alizarin red staining and oil red staining,respectively. In addition,the mRNA expression of osteoblast specific gene and adipocyte specific genes increased obviously after induction. Conclusion:Our research successfully established a culture method for primary mesenchymal stem cells from neonatal mouse calvaria. This method might be important for further studies of mesenchymal stem cells.