人参皂苷Rb1通过SDF-1/CXCR4通路调节牙周膜干细胞增殖及成骨分化

Ginsenoside Rb1 regulates proliferation and osteogenic differentiation of periodontal ligament stem cells by SDF-1/CXCR4 signaling pathway

目的观察人参皂苷Rb1(Gs Rb1)对人牙周膜干细胞(h PDLSCs)增殖和成骨分化的影响,及其与基质细胞衍生因子1(SDF-1)/CXC趋化因子受体4(CXCR4)通路相关分子机制。方法采用酶消化法分离、培养h PDLSCs,倒置显微镜下观察细胞形态,流式细胞术检测基质细胞抗原1(STRO-1)、分化簇146(CD146)阳性细胞比例;分别用0、0.5、1.0、2.0、4.0、6.0μmol·L^(-1)GsRb1处理h PDLSCs,CCK-8法筛选最佳作用浓度。将h PDLSCs分为对照组、Gs Rb1(4.0μmol·L^(-1))组、AMD3100(5μg·m L^(-1))组、Gs Rb1+AMD3100组,比较各组细胞增殖活力和碱性磷酸酶(ALP)活性,RT-q PCR检测Runt相关基因2(Runx2)、oxterix、骨桥蛋白(OPN)m RNA表达情况,茜素红染色、定量分析检测h PDLSCs中矿化结节形成量,Western blot法检测SDF-1/CXCR4信号通路相关蛋白表达情况。结果h PDLSCs呈放射状排列,形态为长梭形,生长较为致密,STRO-1、CD146阳性细胞比例分别为97.19%、98.01%。随着Gs Rb1浓度的增加,h PDLSCs增殖活力增强,呈浓度依赖性(P<0.05)。与对照组比较,Gs Rb1组细胞增殖活力、ALP活性增强,runx2、oxterix、OPN mRNA表达增加,矿化结节形成量及SDF-1、CXCR4蛋白表达均增加(P<0.05),而AMD3100组与之相反(P<0.05)。与Gs Rb1组比较,Gs Rb1+AMD3100组细胞增殖活力、ALP活性降低,Runx2、oxterix、OPN m RNA表达降低,矿化结节形成量及SDF-1、CXCR4蛋白表达均减少(P<0.05)。结论Gs Rb1能够促进h PDLSCs增殖及成骨分化,可能与增加SDF-1/CXCR4信号通路蛋白表达有关。

AIM To observe the influences of ginsenoside Rb1(GsRb1)on the proliferation and osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs),and its molecular mechanism related to stromal cellderived factor-1(SDF-1)/CXC chemokine receptor 4(CXCR4)pathway.METHODS hPDLSCs were isolated and cultured by enzyme digestion method.Cell morphology was observed under an inverted microscope.Flow cytometry was used to detect the proportion of stromal cell antigen 1(STRO-1)and cluster of differentiation 146(CD146)positive cells.hPDLSCs were treated with 0,0.5,1.0,2.0,4.0,6.0μmol·L^(-1)GsRb1,respectively,and the optimal concentration was screened by CCK-8 method.hPDLSCs were divided into control group,GsRb1(4.0μmol·L^(-1))group,AMD3100(5μg·mL^(-1))group,and GsRb1+AMD3100 group,the cell proliferation and alkaline phosphatase(ALP)activity of each group were compared,the mRNA expression of Runt-related gene 2(Runx2),oxterix,and osteopontin(OPN)were detected by qRT-PCR,and the formation of mineralized nodules in hPDLSCs was detected by alizarin red staining and quantitative analysis.Western blot was used to detect the expression of SDF-1/CXCR4 signaling pathway-related proteins.RESULTS hPDLSCs were arranged radially,with long spindles,and the growth was relatively dense.The proportions of STRO-1 and CD146 positive cells were 97.19%and 98.01%,respectively.With the increase of GsRb1 concentration,the proliferation activity of hPDLSCs was enhanced in a dose-dependent manner(P<0.05).Compared with the control group,the cell viability and ALP activity in the GsRb1 group were enhanced,and the mRNA expression of Runx2,oxterix,OPN,the amount of mineralized nodules,and the protein expression of SDF-1 and CXCR4 were increased(P<0.05),while the AMD3100 group was the opposite(P<0.05).Compared with the GsRb1 group,the cell viability and ALP activity in the GsRb1+AMD3100 group were decreased,and the mRNA expression of Runx2,oxterix,OPN,the amount of mineralized nodules,and the protein expression of SDF-1 and CXCR4 were decreased(P<0.05).CONCLUSION GsRb1 can promote the proliferation and osteogenic differentiation of hPDLSCs,which may be related to the increased protein expression of SDF-1/CXCR4 signaling pathway.