钙结合蛋白S100A8/A9阻滞剂对人子宫内膜异位症在位内膜间质细胞系hEM15A的作用效果

Effect of calcium binding protein S100A8/A9 blocker on eutopic endometrial stromal cell line hEM15A in human endometriosis

目的探讨S100钙结合蛋白A8/A9(S100 calcium binding protein A8/A9,S100A8/A9)阻滞剂对异位子宫内膜细胞的作用效果。方法以不同浓度的S100A8/A9蛋白的特异性阻滞剂帕奎莫德(ABR-25757)作用异位子宫内膜细胞(hEM15A)24 h后,通过细胞计数试剂盒-8(CCK8)法检测ABR-25757作用后细胞形态及细胞增殖活力,从4个梯度浓度中筛选出ABR-25757作用的最佳浓度。分为实验组(hEM15A+ABR-25757)和对照组(hEM15A+同体积PBS)。最佳浓度的ABR-25757作用hEM15A细胞24 h后,采用Transwell实验检测细胞的迁移和侵袭能力,采用实时荧光定量聚合酶链反应(real-time PCR,qPCR)检测细胞血管内皮生长因子(VEGF)及转化生长因子β1(TGF-β1)表达水平,采用酶联免疫吸附试验(ELISA)方法检测细胞培养上清液VEGF及TGF-β1水平。结果(1)以100、200、400、800μg/mL等不同浓度梯度ABR-25757作用hEM15A细胞24 h后,与对照组相比,不同浓度ABR-25757组均可降低hEM15A细胞增殖活力(100μg/mL组:P<0.05;其余浓度组:P<0.01)。由于200μg/mL浓度作用效果明显,且细胞死亡少,故选择200μg/mL为最佳浓度;(2)ABR-25757作用细胞后与对照组相比,hEM15A细胞迁移和侵袭能力明显减弱(P<0.01);(3)ABR-25757作用hEM15A细胞后VEGF和TGF-β1的mRNA表达水平均低于对照组(VEGF:P<0.01,TGF-β1:P<0.05);(4)ABR-25757作用后细胞培养上清液VEGF和TGF-β1水平均低于对照组(VEGF:P<0.01,TGF-β1:P<0.05)。结论ABR-25757是S100A8/A9的一种特异性抑制剂,能有效抑制异位内膜细胞的增殖、降低异位内膜细胞迁移和侵袭能力,以及有效下调VEGF、TGF-β1的表达。

Objective To investigate the effect of S100A8/A9 protein blocker on ectopic endometrial cells.Methods Heterotopic endometrial cells(hEM15A)were treated with different concentrations of S100A8/A9 protein blocker Paquimide(ABR-25757)for 24 h,and cell morphology and cell proliferation activity were detected by cell counting kit 8(CCK8)method.The optimal concentration of ABR-25757 was selected from four gradient concentrations.They were divided into experimental group(hEM15A+ABR-25757)and control group(hEM15A+same volume PBS).After 24 h treatment of hEM15A cells with optimal concentration of ABR-25757,the migration and invasion ability of HEM15A cells were detected by Transwell assay.The expression levels of vascular endothelial growth factor(VEGF)and transforming growth factorβ1(TGF-β1)were detected by real-time quantitative polymerase chain reaction(qPCR),and VEGF and TGF-β1 levels were detected by enzyme-linked immunosorbent assay(ELISA).Results①Treated with different concentration(100,200,400,800μg/mL)of ABR-25757 on hEM15A cells for 24 h,cell morphology and proliferation were observed.Compared with the control,different concentrations of ABR-25757 could significantly reduce the proliferation activity of hEM15A cells(100μg/mL group:P<0.05,other concentration groups:P<0.01).200μg/mL concentration was selected to be the best concentration for obvious effect and less cell death.②Compared with the control,the migration and invasion ability of hEM15A cells decreased significantly treated with ABR-25757(P<0.01).③mRNA expression level of VEGF and TGF-β1 in hEM15A cells treated with ABR-25757 were lower than that of the control group(VEGF:P<0.01,TGF-β1:P<0.05).④The level of VEGF and TGF-β1 in cell culture supernatant of hEM15A cells treated with ABR-25757 were lower than that of the control group(VEGF:P<0.01,TGF-β1:P<0.05).Conclusions ABR-25757 is a specific inhibitor of S100A8/A9,which can effectively inhibit the proliferation of ectopic endometrial cells,reduce the migration and invasion ability of ectopic endometrial cells,and effectively down-regulate the expression of VEGF and TGF-β1.