HtrA1及其相关因子MGP、BMP-2在人牙周膜细胞体外成骨分化和矿化过程中的动态表达

Dynamic Expression during Differentiation and Mineralization of Bone HtrA1 and Its Related Factors MGP,BMP-2 in Human Periodontal Ligament Cells in Vitro

目的研究Htr A1、MGP、BMP-2在人牙周膜细胞成骨分化中的生物学作用,初步探讨Htr A1与MGP、BMP-2在这一过程中的表达关系.方法首先用矿化诱导剂(β-磷酸甘油钠、地塞米松和抗坏血酸等)对人牙周膜细胞进行矿化诱导,建立体外牙周膜细胞矿化模型.然后通过检测碱性磷酸酶(ALP)活性,茜素红染色检测矿化结节形成,o-CPC法检测胞外基质钙沉积,以及用荧光定量PCR方法检测成骨分化指标ALP、骨唾液酸蛋白(bone sialoprotein,BSP)、骨钙素(osteocalcin,OCN)、胶原蛋白(COLI)和核心结合蛋白因子2(runt-related transcription factor-2,RUNX2),来证实人牙周膜细胞成功进行了成骨分化以及体外矿化模型成功建立.采用荧光定量PCR和Western Blotting的方法检测Htr A1与MGP、BMP-2的动态表达,研究3者的表达变化规律和相互表达关系.结果人牙周膜细胞在矿化诱导组中的ALP活性显著升高;从矿化诱导的第7天到第14天,可见钙沉积的数量出现陡增,而后钙含量增加则趋于平缓,说明在这一阶段牙周膜细胞发生了较为明显的成骨分化特征改变.而在对照组中,细胞外基质中的钙沉积没有明显变化,与矿化诱导组相比显著降低(P<0.05);在矿化诱导的第0、3、7、11、14、21、28天中,Htr A1蛋白水平表达则呈现逐步升高趋势,而MGP和BMP-2的蛋白表达模式相似,呈现整体逐渐下降的趋势.结论对人牙周膜细胞进行体外成骨诱导的过程中,发现Htr A1的表达水平增高,而其相关信号分子MGP、BMP-2的表达则呈现下降趋势,推测Htr A1、MGP、BMP-2三者共同参与了人牙周膜细胞成骨分化过程,并且Htr A1可能通过调节MGP、BMP-2来发挥作用.

Objective To explore the biological role of Htr A1,MGP,BMP- 2 in osteogenic differentiation in human periodontal ligament cells, and preliminarily study the relationship between the expression of Htr A1 and MGP,BMP- 2 in this process.Methods First of all, mineralization inducing agents（beta glycerophosphate sodium, dexamethasone and ascorbic acid） were used on human periodontal ligament cells in vitro to induce mineralization,so as to establish mineralization of dental periodontal membrane cell model. And then through the detection of alkaline phosphatase（ALP） activity,alizarin red staining and mineralized nodules were detected,the o- CPC method was used to detect the extracellular calcium deposition, and the detection of osteogenic differentiation indicators ALP,BSP,OCN,COLI and Runx2 by fluorescence quantitative PCR method,to confirm the human periodontal ligament cells successfully in vitro osteogenic differentiation and mineralization model was successfully established. Dynamic expression of Htr A1 and MGP, BMP- 2 was detected by fluorescence quantitative PCR and Western Blotting, the relationship between the expression and interaction of the three factors were also analyzed. Results In the induction group, the activity of ALP was significantly increased in the mineralization group of human periodontal ligament cells. From seventh days to fourteenth days after mineralization induction, the number of visible calcium deposition appeared sharply, and calcium content increased slowly, suggesting that periodontal ligament cells had obvious osteogenic differentiation characteristics in this stage. In the control group,the calcium deposition in the extracellular matrix did not change significantly, and was significantly decreased as compared with the mineralization induced group（P〈0.05）. On the 0, 3, 7, 11, 14, 21, 28 days after mineralization induction, the expression level of Htr A1 protein showed a gradually rising trend, while the MGP and BMP- 2 were in a similar expression patterns, showing the overall trend of gradual decline.Conclusion During bone induction of human periodontal ligament cells in vitro into bone induction, the expression level of Htr A1 was increased,and the expression levels of its related signal molecules MGP,BMP- 2 were decreased,suggesting that Htr A1,MGP,BMP- 2 play roles in human periodontal ligament cells osteogenic differentiation process, and Htr A1 may paly roles by the regulation of MGP, BMP- 2.