论文部分内容阅读
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。
Objective: To explore the method of primary culture of neonatal mouse calvariae mesenchymal stem cells. Methods: The calvaria of neonatal mice were harvested and the mesenchymal stem cells were obtained by collagenase digestion. The morphology and proliferation of the cells were observed. The cell proliferation was analyzed by cell cycle. The cell purity was analyzed by flow cytometry. The osteogenic and adipogenic differentiation was induced. Alizarin red and oil red O staining were performed respectively. The osteogenic marker gene Osteocalcin and adipogenic marker genes PPARγ and Fabp4 were detected by fluorescence quantitative PCR. Results: The cultured spindle cells had good homogeneity and strong proliferative ability. Flow cytometry cell surface markers showed good cell purity, high expression of CD29, CD44, almost no expression of CD34, CD45. After differentiation, the differentiation rate was high. Alizarin red staining and oil red O staining showed more mineralized nodules and lipid droplets. Fluorescent quantitative PCR showed that the genes associated with differentiation were significantly increased. Conclusion: The primary culture model of neonatal mouse calvarial stem cells was successfully established in this study, which laid the foundation for further study of mesenchymal stem cells.