高糖和bFGF对骨髓间充质干细胞成骨分化的影响

目的：骨组织的形成是一个复杂的过程，受多种因素的影响，糖尿病所导致的持续高血糖对于成骨分化的影响机制尚不明确，以及在此分化过程中的各种细胞因子的作用机理仍不明了，现拟通过体外成骨诱导环境，观察高糖和碱性成纤维细胞生长因子（fibroblastgrowthfactorbFGF）对人骨髓间充质干细胞（humanmesenchymalstemcellshMSCs）成骨分化的影响。方法：hMSC在5．5mmol／L和25mmol／L葡萄糖浓度下培养6天，使用cck一8法测定各组细胞增殖情况；hMSC在两种糖浓度下成骨诱导28天，通过碱性磷酸酶（ALP）活性检测、茜素红染色、钙结节半定量检测，对比各组成骨分化活性；在两种糖浓度成骨诱导液中加入10ng／mlbFGF，使用RT—PCR技术检测各组细胞OCN、OPNmRNA表达差异。结果：高糖较正常糖浓度细胞增殖率下降，ALP活性降低，茜素红染色钙结节量减少，RT—PCR检测结果显示25mmol／L组OCN、OPNmRNA表达量低于5．5mmol／L组，加入bFGF后，25mmol／L组仍低于5．5mmol／L组，与未添加bFGF同葡萄糖组比较表达增加。结论：高糖使hMSC增殖能力下降，在成骨分化的过程中ALP活性降低，成骨相关基因OCN、OPN表达量下降，证明了高糖对hMSC成骨分化具有抑制作用，当加入bFGF后，改善了高糖对hMSC的抑制作用，提示糖尿病条件下高糖的存在是导致hMSC成骨分化能力下降的不利因素，同时初步证明了bFGF参与了成骨分化的过程，从而为在分子水平探讨糖尿病患者种植义齿骨结合形成相关机制奠定初步的基础．．

Effect of High Glucose and bFGF on the Osteoblastic Differentiation of Bone Marrow Stromal Cells

Objective： Bone tissue formation is a complicated process affected by many factors, the effect of high glucose resulting from diabetes on the osteogenetic differentiation mechanism has not been clear up.We established a model of osteogenic differentiation with BMSCs. To observe the effect of high glucose and basic fibroblast growth factor （bFGF） on osteoblastic differentiation ability in hu- man marrow mesenchymal stem cells （hMSCs）. Methods： HMSC cluture in 5.5 mmol/L and 25 mmol/L glucose concentrations for 6 days, Cell proliferation was assessed by cell counting kit （CCK）-8, HMSC cultured in different concentrations of glucose （5.5, 25 mmol/L） osteogenic medium for 28 days. The effects of high glucose on alkaline phosphatase （ALP） activity, alizarin red dye, calcium nodules half quantitative detection were measured, moreover, add 10 ng/ml bFGF in each groups, RT - PCR detection osteogenesis -related gene OCN, OPN mRNA expression. Results： At higher concentrations of glucose, cellular proliferation, ALP activity, calcium deposition were greatly reduced. Compared with 5.5 mmol/1 normal glucose, OCN, OPN, mRNA expression were greatly reduced in High glucose with or without 10 ng/ml bFGF. Conclusion： High glucose inhibits Cell proliferation ability in BMSCs. ALP activity, osteogenesis -related gene OCN, OPN and Runx2 mRNA expression are reduced, proved that the high glucose has inhibitory effect on hMSC osteogenetic differentiation, BFGF alleviates the inhibitory effects of high glucose on the osteoblastic differentiation of human bone marrow stromal cells. It pointed that the bFGF participated in the osteogenetic differentiation process. To provide the experimental evidences for dental implant in patients with diabetes from molecular level.