由cAMP激活的鸟苷酸交换蛋白(Epac1)调控内皮细胞表面膜联蛋白A2的表达

Exchange protein activated by cAMP(Epac1)regulating the expression of Annexin A2 on endothelial cells surface

目的本研究旨在探讨内皮细胞内Epac1是否参与调控细胞表面膜联蛋白A2(ANXA2)的表达.方法以人静脉内皮细胞(HUVECs)为细胞模型,采用原子动力学检测方法,结合免疫荧光,分别从原子及蛋白水平验证Epac1是否参与细胞表面ANXA2表达的调控作用.分别提取15只Epac1基因敲除小鼠和15只C57BL6野生型小鼠血浆,检测每组样品ANXA2的表达水平.结果①免疫荧光测得对照组(ESI09)相对荧光强度为17.59±0.69,处理组(ESI09)相对荧光强度为6.32±0.32,两组间数据结果有差异且有统计学意义(P<0.01);原子动力显微镜法测得处理组(ESI09)测得力值为(35113.07±9866.65)pN,对照组(DMSO)测得力值为(106277.70±14233.77)pN,两组间数据结果有差异且有统计学意义(P<0.01);由以上数据可知药物性抑制内皮细胞Epac的功能使其表面ANXA2表达量下调;②蛋白印迹法及酶联免疫吸附法测得处理组(ESI09)和对照组(DMSO)内皮细胞分泌ANXA2有差异,药物性抑制内皮细胞Epac1功能使内皮细胞胞外分泌ANXA2能力下降低;③酶联免疫吸附法测得Epac1基因敲除小鼠组血浆ANXA2平均含量为(138.81±38.93)ng/ml,显著低于野生型小鼠组血浆ANXA2平均含量(281.46±49.66)ng/ml(P<0.05).结论Epacl参与调控内皮细胞表面ANXA2的表达.

Objective To explore the role of Epacl in regulating endothelial surface expression of ANXA2.Methods making HUVECs as cell model,Novel technique atomic force microscopy(AFM)was applied to explore ANXA2 on ECs surface,confirmed with immunofluorescence(IF).Enzyme linked immunosorbent assay(ELISA)was used to determine ANXA2 concentration in plasma of Epacl-null mice(n=15)compared with that of wild-type mice(n=15).Results(1)Pharmacologically functional suppression of Epacl in HUVECs via ESI09 decreased ANXA2 residing on EC apical surfaces:relative IF intensity showed difference between ESI09-treated group and DMSO-treated group(6.32±0.32 vs.17.59±0.69,P<0.01);AFM force measurement also shows difference between ESI09-treated group and DMSO-treated group[(106277.70土14233.77)pN vs.(35113.07±9866.65)pN,P<0.01].(2)Both WB and ELISA showed pharmacologically functional suppression of Epacl in HUVECs via ESI09 decreases ANXA2 secretion.(3)Depletion of Epacl resulted in decreased ANXA2 in mice plasma[(138.81±38.93)ng/ml in plasma of Epacl-null mice vs.(281.46±49.66)ng/ml in plasma of wild-type mice,P<0.05].Conclusion Epacl participates in regulating the expression of ANXA2 on ECs.