爱帕琳肽13/血管紧张素受体AT1相关受体蛋白同源二聚体对人脐静脉内皮细胞行为的影响

Effects of Apelin-13/putative receptor protein related to AT1 homodimer on behaviors of human umbilical vein endothelial cells

目的探究血管紧张素受体AT1相关受体蛋白(putative receptor protein related to AT1,APJ)同源二聚体对人脐静脉内皮细胞(HUVECs)行为,即增殖、迁移和成管的作用。方法将HUVECs随机分为对照组(PBS)、APJ单体组[爱帕琳肽13(Apelin-13)+TM1]和APJ同源二聚体组(Apelin-13+PBS),分别用Western blot和基质辅助激光解吸飞行时间质谱仪(MALDI-TOF MS)检测APJ和APJ同源二聚体在HUVECs中的表达;实时细胞分析仪(RTCA)检测Apelin-13引起50%最大效应的浓度(EC_(50));CCK-8法测定HUVECs的细胞活力;划痕实验检测HUVECs的迁移能力;人工基底膜(matrigel)检测HUVECs的成管。结果Western blot和MALDI-TOF MS实验结果显示,APJ和APJ同源二聚体均表达于HUVECs中。Apelin-13的EC_(50)为2.26×10^(-8)mol·L^(-1),且引起最大效应的浓度为1.0×10^(-6)mol·L^(-1)。CCK-8实验、迁移实验和成管实验结果显示,各组细胞随着时间的延长逐渐向划痕裸区迁移,镜下也观察到各组细胞均出现成管,数据统计发现Apelin-13+PBS和Apelin-13+TM1组的HUVECs增殖、迁移和成管能力均较PBS组明显增加,且Apelin-13+PBS组的增殖、迁移和成管能力明显好于Apelin-13+TM1组(P<0.05)。结论APJ同源二聚体对HUVECs的增殖、迁移和成管均具有促进作用,且效果优于APJ单体。

Aim To explore the effects of putative receptor protein related to AT1(APJ)homodimer on the behaviors-the proliferation,migration and tube formation of human umbilical vein endothelial cells(HUVECs).Methods HUVECs at logarithmic growth stage were randomly divided into PBS,Apelin-13+TM1(APJ monomer group)and Apelin-13+PBS group(APJ homodimer group).Western blot and Matrix-Assisted Laser Desorption/Ionization Time of Fligh Mass Spectrometry(MALDI-TOF MS)were used to detect the expression of APJ and APJ homodimer in HUVECs,respectively.Real-Time Cell Analyzers(RTCA)was used to detect the concentration of the maximum effect of Apelin-13.Cell viability was detected by CCK-8.The cell migration ability was detected by scratch test,and the number of tubes formed on matrigel that made artificial basement membrane was counted.Results Western blot and MALDI-TOF MS showed that APJ and APJ homodimer were expressed in HUVECs.The EC 50 of Apelin-13 was 2.26×10^(-8)mol·L^(-1),and the concentration of the maximum effect was 1.0×10^(-6)mol·L^(-1).The results of CCK-8 experiment,migration experiment and tube formation showed that cells in each group migrated to the scratch bare area gradually with the extension of time,and the tube formation of cells in each group was also observed under the microscope.Data statistics showed that the proliferation,migration and tube forming abilities of HUVECs in Apelin-13+PBS and Apelin-13+TM1 groups were significantly higher than those in PBS group,and the abilities of proliferation,migration and tube forming in Apelin-13+PBS group were significantly better than those in Apelin-13+TM1 group(P<0.05).Conclusions APJ homodimer can promote the proliferation,migration and tube formation of HUVECs,and the effects are better than APJ monomer.