PDGFRβ-PI3K信号轴介导骨折愈合过程中骨膜细胞激活的分子机制研究

Molecular mechanism of PDGFRβ-PI3K signaling axis mediating periosteal cell activation during fracture healing

目的验证血小板源性生长因子受体β/磷酸肌醇3-激酶(PDGFRβ-PI3K)相互作用对于介导骨折后强大的骨膜细胞活化是否有必要。方法收集正常活动或骨折手术后3 d的小鼠骨膜细胞进行体外原代培养,分为空白组、阴性组、shPDGFβ组和各预处理组,空白组不做任何处理,阴性组和shPDGFβ组分别转染对照shRNA和PDGFRβshRNA慢病毒,72 h后用于实验,各预处理组包括用生长因子(PDGF-BB,10 ng/mL)、PDGFR抑制剂(SU16f,5μmol/L)、小分子AKT激活剂(SC79,4μg/mL)或PI3K激活剂(740 Y-P,25 nmol/L)单独或序贯处理30 min。采用Western blot试验、流式细胞术、EdU染色实验检测各组细胞蛋白表达水平和增殖活力。结果与正常活动小鼠的骨膜细胞比较,骨折小鼠的骨膜细胞中PDGFRβ蛋白表达水平升高约2.31倍。通过流式细胞术验证,骨折小鼠骨膜细胞中PDGFRβ阳性率为(73.26±3.17)%。在骨折小鼠的骨膜细胞实验中,shPDGFRβ组细胞p-AKT蛋白表达水平均低于空白组和阴性组,差异均有统计学意义(P<0.05);而PDGF-BB组和SC79组细胞p-AKT蛋白表达水平一致,差异无统计学意义(P>0.05)。与空白组(PI3K:1.22±0.09,p-Tyr-PDGFRβ:0.01±0.01)比较,PI3K激活剂740 Y-P组可上调PI3K水平(1.67±0.13)和p-Tyr-PDGFRβ水平(0.07±0.01),差异均有统计学意义(P<0.05),但对PDGFβ水平无明显干扰。另外,与740 Y-P组[p-AKT(Ser473):0.32±0.03]比较,740 Y-P+PDGF-BB组进一步上调了骨膜细胞中p-Tyr-PDGFRβ水平(0.19±0.01)和p-AKT(Ser473)水平(0.51±0.05),差异均有统计学意义(P<0.05)。在体外增殖实验中,没有对于用740 Y-P预处理的细胞,加入PDGF-BB后,细胞增殖活性几乎恢复至阴性组水平,明显高于未用740 Y-P预处理的细胞。结论PDGFRβ-PI3K信号轴对于骨折愈合早期阶段的骨膜活化是有必要的。

Objective To verify whether platelet-derived growth factor receptorβ/phosphatidylinositol 3-kinase(PDGFRβ-PI3K)interaction is necessary for mediating robust periosteal cell activation after fracture.Methods Periosteal cells were collected from mice with normal activity or 3 days after fracture surgery for primary culture in vitro.The cells were divided into blank group,negative group,shPDGFRβgroup and each pretreatment group.The blank group did not receive any treatment,while the negative group and shPDGFRβgroup were transfected with control shRNA and PDGFRβshRNA lentivirus respectively,and used for the experiment after 72 hours.The pretreatment groups included growth factor(PDGF-BB,10 ng/mL),PDGFR inhibitor(SU16f,5μmol/L),small molecule AKT activator(SC79,4μg/mL)or PI3K activator(740 Y-P,25 nmol/L)alone or sequentially treated for 30 min.Western blot assay,flow cytometry,and EdU staining were used to detect the protein expression level and proliferation activity of cells in each group.Results Compared with the periosteal cells of normal active mice,the expression level of PDGFRβprotein in the periosteal cells of fracture mice was increased by about 2.31 times.The positive rate of PDGFRβin periosteal cells of fracture mice was(73.26±3.17)%verified by flow cytometry.In the periosteal cell experiment of fracture mice,the expression level of p-Akt protein in shPDGFRβgroup was lower than that in the blank group and the negative group,and the difference was statistically significant(P<0.05).However,there was no significant difference in the expression of p-Akt protein between PDGF-BB group and SC79 group(P>0.05).Compared with the blank group(PI3K:1.22±0.09,p-Tyr-PDGFRβ:0.01±0.01),the PI3K activator 740 Y-P group could increase the level of PI3K(1.67±0.13)and p-Tyr-PDGFRβ(0.07±0.01),and the differences were statistically significant(P<0.05),but there was no significant interference on the level of PDGFRβ.In addition,compared with the 740 Y-P group[p-AKT(Ser473):0.32±0.03],the 740 Y-P+PDGF-BB group further increased p-Tyr-PDGFRβlevel(0.19±0.01)and p-Akt(Ser473)level(0.51±0.05)in the periosteal cells,and the differences were statistically significant(P<0.05).In the in vitro proliferation experiment,the proliferation activity of the cells pretreated with 740 Y-P was almost restored to the level of the negative group after adding PDGF-BB,which was significantly higher than that of the cells without 740 Y-P pretreatment.Conclusion PDGFR beta PI3K signaling shaft for fracture healing in the early stages of periosteum activation is necessary.