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目的:探讨胰蛋白酶对人脐静脉内皮细胞(HUVECs)分泌内皮素(ET)-1的影响。方法:分离、培养HUVECs,细胞免疫荧光技术检测内皮细胞表达蛋白酶活化受体(PAR)-2。内皮细胞生长添加物和肝素培养体系培养HUVECs,激活剂为胰蛋白酶和丝-亮-异亮-甘-赖-缬(SLIGKV-NH2),ELISA检测细胞上清中ET-1水平。结果:FITC标记的抗人PAR-2抗体染色细胞的荧光主要存在于细胞周边及突起处,说明HUVECs表达PAR-2。随着胰蛋白酶和SLIGKV-NH2浓度升高(与单纯细胞培养液比,P<0.05),HUVECs分泌ET-1的水平也相应升高。两种刺激剂诱导的ET-1水平正相关。α1-抗胰蛋白酶、大豆胰蛋白酶抑制剂和PAR-2抑制肽显著抑制胰蛋白酶或SLIGKV-NH2诱导的ET-1释放。结论:胰蛋白酶很可能通过PAR-2促进内皮细胞分泌ET-1。
Objective: To investigate the effect of trypsin on the secretion of endothelin (ET) -1 from human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were isolated and cultured. The expression of protease - activated receptor (PAR) -2 in endothelial cells was detected by immunofluorescence staining. HUVECs were cultured with endothelial cell growth supplement and heparin culture system. The activators were trypsin and SLIGKV-NH2, and the levels of ET-1 in the supernatant were detected by ELISA. Results: The fluorescence of FITC-labeled anti-human PAR-2 antibody staining cells mainly in the periphery and protrusions, indicating that PAR-2 was expressed in HUVECs. With the increase of trypsin and SLIGKV-NH2 concentration (compared with the simple cell culture medium, P <0.05), the level of ET-1 secreted by HUVECs increased accordingly. The levels of ET-1 induced by the two stimulants were positively correlated. Alpha 1 -antitrypsin, soy trypsin inhibitor, and PAR-2 inhibitory peptide significantly inhibited trypsin or SLIGKV-NH2-induced ET-1 release. Conclusion: Trypsin may promote the secretion of ET-1 by endothelial cells through PAR-2.