p53在层流剪切应力下对EPCs分化及功能的影响

Role of p53 in differentiation and function of EPCs under laminar shear stress

目的:研究p53蛋白在层流剪切应力(LSS;12 dyn/cm^(2))作用下对内皮祖细胞(EPCs)分化及功能的影响,为心血管疾病的防治找到新的靶点。方法:采用密度梯度离心法获得大鼠骨髓单个核细胞,用EGM-2培养基诱导获得EPCs;通过Western blot方法检测p-p53及p53蛋白水平的变化;采用RT-qPCR及流式细胞术分别检测内皮标志物CD31和vWF的mRNA及蛋白表达;使用ELISA检测培养液上清中vWF含量;分别利用Matrigel、Boyden小室、细胞贴壁法、CCK-8法和EdU技术检测EPCs成血管、迁移、黏附和增殖能力。结果:LSS作用促进EPCs中pp53和p53蛋白水平升高;p53抑制剂pifithrin-α处理后,与单纯LSS组相比,EPCs的CD31和vWF表达、黏附、迁移及成血管能力均降低,但其增殖能力升高。结论:LSS是调节EPCs分化及其生物学功能的重要力学因素,而p53蛋白可能在该过程中起关键的调节作用。

AIM:To study the effect of laminar shear stress(LSS)on the differentiation and function of endothelial progenitor cells(EPCs)and the role of p53 in this process,so as to discover the new targets for the prevention and treatment of vascular remodeling-related diseases.METHODS:Rat mononuclear bone marrow cells were obtained by density gradient centrifugation and were cultured with EGM-2 medium.The protein levels of p-p53 and p53 were determined by Western blot.RT-qPCR and flow cytometry were used to test the expression of endothelial marker CD31 and vWF.The vWF protein secreted in supernatant was measured by ELISA.The tube formation,migration,adhesion and proliferation of EPCs were checked by Matrigel,Boyden chamber,cell attachment,CCK-8 and EdU assays,respectively.RESULTS:The protein levels of p-p53 and p53 were increased after LSS on EPCs.Compared with LSS group,the expression of CD31 and vWF in LSS+pifithrin-α group was decreased,the abilities of adhesion,migration and angiogenesis were reduced,but the proliferation was increased.CONCLUSION:Laminar shear stress is an important mechanical factor to regulate the differentiation and biological function of EPCs,and p53 may play a vital role in this process.