重组人HMGB1调控内皮细胞成血管作用的机制初探

Mechanism of regulatory effect of recombinant human HMGB1 on endothelial cell angiogenesis

目的探讨重组人高迁移率族蛋白1(rhHMGB1)在调控内皮细胞成血管过程中可能涉及的机制。方法将内皮细胞分为对照组、骨髓间充质干细胞(MSC)上清组以及rhHMGB1组。采用细胞计数试剂(CCK)-8法检测内皮细胞的增殖、存活情况;采用蛋白质印迹法检测血管内皮生长因子(VEGF)、Yes相关蛋白(YAP)、CD31、缺氧诱导因子(HIF)-1α的蛋白相对表达量;采用实时荧光定量聚合酶链反应(RT-qPCR)检测VEGF、YAP、CD31、HIF-1α的信使RNA(mRNA)相对表达量;采用Transwell实验检测内皮细胞迁移水平;免疫荧光实验检测YAP定位情况。结果与对照组比较,rhHMGB1组内皮细胞迁移率增加(P<0.05),且细胞迁移率随着时间的延长而增加。与对照组比较,MSC上清组VEGF蛋白相对表达量增加,p-YAP蛋白表达增多,差异均有统计学意义(均为P<0.05)。与对照组比较,rhHMGB1组的VEGF和HIF-1α蛋白相对表达量以及VEGF和CD31 mRNA相对表达量均增加,YAP和p-YAP蛋白表达均增多,YAP/p-YAP比值增加,差异均有统计学意义(均为P<0.05)。与MSC上清组比较,rhHMGB1组的CD31 mRNA相对表达量增加,YAP蛋白表达增多,YAP/p-YAP比值增加,差异均有统计学意义(均为P<0.05)。结论外源高浓度rhHMGB1可能通过介导YAP入核促进内皮细胞的迁移能力,并上调血管生成相关蛋白的表达。

Objective To unravel the possible mechanism of the role of recombinant human high mobility group box 1(rhHMGB1)protein in regulating the angiogenesis of endothelial cells.Methods Endothelial cells were divided into the control group,bone marrow mesenchymal stem cells(MSC)supernatant group and rhHMGB1 group.The proliferation and survival of endothelial cells were detected by cell counting kit(CCK)-8 assay.The relative expression levels of vascular endothelial growth factor(VEGF),Yes-associated protein(YAP),CD31 and hypoxia inducible factor(HIF)-1αproteins were determined by Western blot.The relative expression levels of VEGF,YAP,CD31 and HIF-1αmessenger RNA(mRNA)were detected by real-time fluorescent quantitative polymerase chain reaction(RT-qPCR).The migration ability of endothelial cells was assessed by Transwell chamber test.The localization of YAP was detected by immunofluorescence staining.Results Compared with the control group,the migration rate of endothelial cells was increased in the rhHMGB1 group(P<0.05),and the cell migration rate was enhanced over time.Compared with the control group,the relative expression levels of VEGF and p-YAP proteins were up-regulated in the MSC supernatant group,and the differences were statistically significant(both P<0.05).Compared with the control group,the relative expression levels of VEGF and HIF-1αproteins,VEGF and CD31 mRNA and YAP and p-YAP proteins were up-regulated,and YAP/p-YAP ratio was increased in the rhHMGB1 group,and the differences were statistically significant(all P<0.05).Compared with the MSC supernatant group,the relative expression levels of CD31 mRNA and YAP protein were up-regulated,and the YAP/p-YAP ratio was increased in the rhHMGB1 group,and the differences were statistically significant(all P<0.05).Conclusions Exogenous high-concentration rhHMGB1 may promote the migration ability of endothelial cells and upregulate the expression levels of angiogenesis-related proteins by regulating the recruitment of YAP to the nucleus.