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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(fibroblast growth factor bFGF)对人骨髓间充质干细胞(human mesenchymal stem cells hMSCs)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-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10 ng/ml bFGF,使用RT-PCR技术检测各组细胞OCN、OPN mRNA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25 mmol/L组OCN、OPN mRNA表达量低于5.5 mmol/L组,加入bFGF后,25 mmol/L组仍低于5.5 mmol/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。
OBJECTIVE: The formation of bone tissue is a complicated process. The mechanism of the effect of persistent hyperglycemia caused by diabetes on osteogenic differentiation is not clear yet, and the role of various cytokines in this differentiation process is influenced by many factors The mechanism is still unknown. It is proposed that the effect of high glucose and fibroblast growth factor bFGF on the osteogenic differentiation of human mesenchymal stem cells hMSCs . Methods: hMSCs were cultured in 5.5mmol / L and 25mmol / L glucose for 6 days. The cell proliferation was determined by cck-8 method. Osteoblasts were induced by hMSC for 28 days at two different concentrations of glucose, ALP) activity assay, alizarin red staining and semi-quantitative detection of calcium nodules were performed to compare the osteogenic activity of each group. 10ng / ml bFGF was added to the osteogenic induction medium of two kinds of sugar concentration, and RT- OCN, OPN mRNA expression differences. Results: Compared with normal glucose, the proliferation rate of high glucose decreased, ALP activity decreased and alizarin red stained calcium decreased. The expression of OCN and OPN mRNA in 25 mmol / L group was lower than 5.5 mmol / L Group, bFGF added, 25 mmol / L group is still lower than 5.5 mmol / L group, with no added bFGF compared with glucose group increased. CONCLUSION: High glucose can decrease the proliferation of hMSC, decrease the activity of ALP during osteogenic differentiation, and decrease the expression of OCN and OPN in osteoblast. It is proved that high glucose can inhibit the osteogenic differentiation of hMSC. When adding bFGF, Improve high glucose inhibition of hMSC, suggesting that the presence of high glucose in diabetic conditions leading to osteogenic differentiation of hMSC decreased adverse factors, and preliminary evidence of bFGF involved in the process of osteogenic differentiation, so as to explore the molecular level of diabetes Patient implant denture bone formation and related mechanisms laid the initial foundation.