葡萄糖、胰岛素对大鼠骨髓间充质干细胞向成骨细胞分化的影响

Effects of glucose and INsulin in the cell differentiation from bone marrow stem cells to osteoblasts

目的观察不同浓度葡萄糖及胰岛素（INs）对骨髓间充质干细胞（BMSC）向成骨细胞（OB）分化能力的影响，探讨葡萄糖及INs对骨代谢的作用机制。方法采用体外细胞培养技术自大鼠股骨和胫骨中分离BMSC进行纯化，培养，然后在成骨培养基中诱导BMSC分化为OB，并用不同浓度的葡萄糖（5．6、25、50mmol／L）及加或不加INs（0．6μg／ml）干预诱导过程，光镜下观察茜素红染色分化后的OB钙结节的细胞比例。RealtimePCR测定OB的标记物碱性磷酸酶（ALP）、骨钙素（BGP）及转录因子Runx2mRNA表达。结果随着糖浓度升高，茜素红染色红色钙结节数目明显减少，PCR结果显示ALP，BGP及Runx2mRNA表达也明显降低，差异有统计学意义（P〈0．05）。提示随着糖浓度增加成骨分化逐渐减少，在同等糖浓度下，加入INs干预组，茜素红染色阳性细胞计数明显增加，PCR结果ALP，BGP及Runx2mRNA表达明显升高（P〈0．01），提示INs可提高OB分化。结论葡萄糖呈量效性地抑制BMSC向OB分化，这可能为糖尿病性骨质疏松形成的重要机制之一。INs可促进BMSC向OB分化，并可改善高糖对BMSC向OB分化的抑制作用。

Objective To observe the effects of different concentrations of glucose and insulin in cell differentiation from bone marrow stem cells （BMSC） into osteoblasts （OB） and explore the acting mechanism of glucose and insulin in bone metabolism. Methods The in vitro technique of cell culture was employed to separate, purify and cultivate BMSC from the tibia and femur of rats. Then the BMSCs were differentiated into osteoblasts under the inductions of different glucose concentrations （5.6, 25,50 retooL/L） with or without the addition of insulin （0. 6 μg/ml）. Later the calcium nodule cell ratio of osteoblasts in alizarin red stain was observed microscopically to assay the mRNA expression of OB markers - ALP, BGP and Runx2 by real-time PCR. Results With the increasing glucose concentrations, the number of red calcium nodules obviously decreased. PCR showed that the mRNA expression of ALP, BGP and transcription factor Runx2 decreased too. The difference was significant （ P 〈 0. 05 ）. The differentiation of OB decreased with with the increasing glucose concentrations. The cell number of red calcium nodules obviously increased in the same glucose concentration groups. The mRNA expression of ALP, BGP and Runx2 also increased （P 〈0. 01）. It hinted that insulin could enhance the differentiation of OB. Conclusion Glucose can inhibit the differentiation from BMSC into OB. The inhibition is more sensitive along with the concentration increments. This may be one of the pathogenesises of diabetic osteoporosis. Insulin can enhance the differentiation from BMSC into OB. In addition, it can improve the inhibition of differentiation from BMSC into OB because of a high concentration of glucose.