炎症微环境下低强度高频振动对牙周膜干细胞成骨分化能力的影响

Effect of LMHFV on Osteogenic Differentiation of Periodontal Ligament Stem Cells in Inflammation Microenvironment

目的:研究在炎症微环境下低强度高频振动(Low-magnitude high frequency vibration,LMHFV)对牙周膜干细胞(Periodontal ligament stem cells,PDLSCs)成骨分化能力的影响。方法:体外分离培养PDLSCs;流式细胞术检测间充质干细胞表面标志物;构建体外炎症微环境:通过向常规培养基(含10%FBS,α-MEM培养基)中添加TNF-α(10ng/ml)与IL-1β(5ng/ml)模拟炎症微环境;加载LMHFV(加速度=0.3g、频率=40Hz,时间=15min/24h)刺激;通过Realtime RTPCR、Western-blot检测成骨相关基因、蛋白表达水平;通过茜素红染色检测细胞成骨分化能力。采用SPSS 21.0软件对数据进行统计学分析。结果:在炎症微环境下,LMHFV刺激后PDLSCs的RUNX-2、ALP、COL-1基因表达量明显升高,ALP、COL-1、OCN的蛋白表达量明显升高,但相较常规培养的PDLSCs,其成骨基因与蛋白表达量仍有明显降低。结论:炎症微环境下,LMHFV刺激可以一定程度上提高PDLSCs的成骨分化能力,但是并不能完全抵消炎性条件对PDLSCs成骨能力的抑制作用。

Objective To investigate the effect of low magnitude high frequency vibration(LMHFV)on osteogenic differentiation of periodontal ligament stem cells(PDLSCs)in an inflammatory microenvironment.Methods PDLSCs were isolated from premolar teeth and mesenchymal stem cell surface markers were analyzed by flow cytometry.Construction of inflammatory microenvironment in vitro:the inflammatory microenvironment was simulated by adding TNF-α(10ng/ml)and IL-1β(5ng/ml)to the conventional medium(containing 10%FBS,α-MEM culture medium).Loading LMHFV stimulation(acceleration=0.3g,frequency=40Hz,time=15min/24h).The expression of osteogenesis gene was analyzed by Realtime RTPCR,and the expression of osteogenesis protein was analyzed by Western-blot.The capability of osteogenesis differentiation was evaluated by alizarin red staining.SPSS 21.0 software was used to analyze the data.Results In the inflammatory microenvironment,the gene expressions of Runx-2,ALP,COL-1 and the protein expressions of ALP,COL-1 and OCN in PDLSCs stimulated by LMHFV were significantly increased.However,compared with the conventional cultured PDLSCs,the osteogenic gene and protein expressions were still significantly decreased.Conclusion LMHFV could enhance osteogenic differentiation of PDLSCs in inflammatory microenvironment.However,it can not completely counteract the inhibitory effect of inflammatory microenvironment on the osteogenic differentiation ability of PDLSCs.