胰岛素促进脐带间充质干细胞成骨分化的作用

摘要

背景：如何有效快速的诱导脐带间充质干细胞成骨分化是当前干细胞研究的重点。既往研究表明生长因子如骨形成蛋白2对脐带间充质干细胞的分化有重要作用，但是生长因子费用较高，不利于普遍使用。胰岛素在细胞培养及诱导过程中广为使用，胰岛素对脐带间充质干细胞有何作用当前未见研究。目的：观察胰岛素对脐带间充质干细胞成骨分化的影响，探讨脐带间充质干细胞治疗糖尿病性骨折愈合延迟的可行性。方法：将第3代人脐带间充质干细胞分别接种于2个细胞培养瓶中，分为实验组与对照组。实验组加入10-7 mmol/L胰岛素的培养液，对照组未加胰岛素培养，采用细胞增殖活性检测试剂盒分别绘制两组细胞的增殖曲线来评价增殖能力，采用细胞碱性磷酸酶活性比色法定量检测碱性磷酸酶的活性，采用免疫细胞化学方法与实时荧光定量聚合酶链反应方法检测Ⅰ型胶原蛋白、mRNA的表达及骨钙素mRNA含量。结果与结论：诱导一两周后，同对照组相比，实验组脐带间充质干细胞的数量增加；胞内碱性磷酸酶的活性提高，Ⅰ型胶原蛋白 mRNA 表达和骨钙素mRNA含量增加(P <0.05)。结果说明胰岛素能够促进脐带间充质干细胞的增殖与成骨分化。%BACKGROUND: How to effectively and rapidly induce the osteogenic differentiation of human umbilical cord mesenchymal stem cells is the focus of the current stem cell research. Increasing evidence has demonstrated some growth factors, such as bone morphogenetic protein-2, have important effects on the transdifferentiation of umbilical cord mesenchymal stem cels into osteoblasts in vitro. However, widespread use of growth factors is limited because of high cost. Insulin is widely used in the cell culture and induction, but there is no report about the effect of insulin on the osteogenic differentiation of human umbilical cord mesenchymal stem cells. OBJECTIVE:To observe the effect of insulin on osteogenic differentiation of human umbilical cord mesenchymal stem cels and to explore the feasibility of human umbilical cord mesenchymal stem cell transplantation in the treatment of diabetic delayed fracture healing. METHODS:The passage 3 human umbilical cord mesenchymal stem cells were inoculated in two flasks, denoted as experimental group and control group. The insulin (10-7mmol/L) was added to the experimental group but not to the control group. The proliferative capacity of human umbilical cord mesenchymal stem cels was evaluated by cell count kit-8 and alkaline phosphatase activity. The osteogenic differentiation capacity of human umbilical cord mesenchymal stem cells was evaluated by measuring the protein and mRNA expressions of type I colagen as well as osteocalcin mRNA level. RESULTS AND CONCLUSION: After 1-2 weeks of induction, compared with the control group, insulin could significantly increase the number of human umbilical cord mesenchymal stem cells in the experimental group, the activity of alkaline phosphatase and expressions of type I collagen osteocalcin mRNA (P< 0.05). These data indicate that insulin can promote the proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells.