镁对猪主动脉瓣间质细胞成骨分化的作用及其机制

Effect and mechanism of magnesium on osteogenic differentiation in porcine aortic valve interstitial cells

目的探究镁对猪主动脉瓣间质细胞(aortic valve interstitial cells,AVICs)成骨分化的调节作用及其潜在机制。方法采用Ⅱ型胶原酶两步消化法分离培养AVICs,采用免疫荧光染色以及流式细胞术进行表型鉴定。将细胞分为空白对照组(Blank组)、成骨诱导培养基组(OIM组)和镁处理组(浓度为1、2、3 mmol/L MgSO_(4)溶液),分别处理7 d。采用Western blot检测各组细胞中成骨分化标志物骨桥蛋白(osteopontin,OPN)、RUNT相关转录因子2(RUNX2)表达量的变化;茜素红染色检测各组细胞中钙盐沉积情况;Western blot检测镁对PI3K/AKT和MAPK/ERK1/2信号通路的作用情况。结果AVICs高表达波形蛋白(Vimentin),低表达α-平滑肌肌动蛋白(α-SMA),不表达CD31,AVICs纯度理想。Western blot结果提示与Blank组相比,OIM组的成骨标志物OPN、RUNX2表达量显著增加(P<0.01);与OIM组相比,3 mmol/L镁处理组可显著降低体外OIM诱导AVICs的OPN和RUNX2表达水平(P<0.01)。茜素红染色可见与Blank组相比,OIM组钙盐沉积明显增加;与OIM组相比,镁处理组能抑制体外OIM诱导的钙盐沉积。Western blot结果提示镁能够显著降低OIM诱导的PI3K/AKT和MAPK/ERK1/2的磷酸化水平(P<0.01)。结论镁可能通过调节PI3K/AKT和MAPK/ERK1/2信号通路,在一定程度上抑制体外OIM诱导AVICs成骨分化的发生。

ObjectiveTo explore the effect of magnesium ion on osteogenic differentiation of porcine aortic valve interstitial cells(AVICs)and its potential mechanism.MethodsPorcine AVICs were isolated from fresh pig aortic valve samples through two-step digestion combined with typeⅡcollagen,and then cultured and identified with immunofluorescence staining and flow cytometry for cell phenotypes.The cells were randomly divided into blank control group(Blank Group),osteogenic induction medium group(OIM Group)and magnesium treatment group(1,2 and 3 mmol/L MgSO_(4) solution for 7 d).The expression of osteogenic differentiation markers,osteopontin(OPN)and Runt-related transcription factor 2(RUNX2)were detected by Western blotting.Alizarin red staining was adopted to observe calcium deposition in the cells of each group.Subsequently,the changes in PI3K/Akt and MAPK/ERK1/2 signaling pathways were measured by Western blotting.ResultsOur obtained porcine AVICs had high expression of Vimentin,low expression ofα-SMA,and no expression of CD31,with an ideal purity of porcine AVICs for subsequent experiments.Western blotting showed that the expression of osteogenic markers OPN and RUNX2 was significantly higher in the OIM Group than the Blank Group(P<0.01),and these effects induced by OIM were obviously inhibited with 3 mmol/L magnesium treatment(P<0.01).Alizarin red staining displayed that calcium salt deposition was remarkably increased in the OIM Group than the Blank Group,and this increment was inhibited after magnesium treatment.The results of Western blotting indicated that magnesium treatment resulted in decreased phosphorylation of PI3K/AKT and MAPK/ERK1/2 induced by OIM(P<0.01).ConclusionMagnesium may partially inhibit the osteogenic differentiation of porcine AVICs induced by OIM in vitro through PI3K/AKT and MAPK/ERK1/2 signaling pathways.