sFlt-1对人单核细胞系THP-1细胞向VEGF迁移的抑制作用

目的 探讨可溶性血管内皮生长因子受体1[sFlt-1(1-3)]对人单核细胞系THP-1细胞向血管内皮生长因子(VEGF)迁移的抑制作用.方法 应用腺病毒包装系统构建携带人sFlt-1(1-3)/FLAG融合基因的重组腺病毒载体(Ad-sFlt-1/FLAG).应用酶联免疫吸附实验(ELISA)和免疫荧光分析所构建的重组腺病毒载体在L293细胞中的表达.通过Transwell实验观察sFlt-1(1-3)对THP-1细胞向VEGF迁移的抑制作用.分别观察5种浓度(0、0.1、1、10和100 ng/mL)的VEGF对THP-1的趋化作用.观察4种浓度(0.1、1、10和100 ng/mL)的sFlt-1(1-3)/FLAG表达上清对THP-1细胞向VEGF迁移的抑制作用.结果 成功构建了Ad-sFlt-1/FLAG融合基因的重组腺病毒载体,通过ELISA和免疫荧光检测显示构建的腺病毒载体携带的sFlt-1(1-3)基因能够在L293细胞中得到表达.在感染重组腺病毒(Ad-sFlt-1/FLAG)的L929细胞培养上清可以检测到sFlt-1(1-3)/FLAG蛋白的表达,浓度为503.7 ng/mL.VEGF终浓度为1、10和100 ng/mL时,迁移的THP-1细胞数高于对照组,差异有统计学意义(P＜0.05).sFlt1(1-3)/FLAG浓度为100 ng/mL时THP-1细胞迁移数约为sFlt1(1-3)/FLAG浓度为0 ng/mL时的25.7%.结论 VEGF对THP-1细胞具有趋化作用,sFlt-1(1-3)可以有效地抑制THP-1细胞向VEGF迁移.

Inhibiting effect of sFlt-1 on migration of human monocytic THP-1 cells in response to VEGF

Objective To investigate the regulation and contribution of vascular endothelial growth factor(VEGF) and sFlt-1(1-3) on human monocytic THP-1 migration.Methods Ad-sFlt-1/FLAG,a recombinant adenovirus carrying the human sFlt-1(1-3) gene was constructed.L929 cells were infected with recombinant adenovirus and then stained for immunofluorescent assay.The expression of sFlt-1(1-3) was detected by enzyme linked immunosorbent assay(ELISA).Transwell Filter Inserts containing PET membranes were used as an experimental model to simulate THP-1 migration.Five VEGF concentrations(0,0.1,1,10 and 100 ng/mL) and four concentrations of sFlt-1(1-3)/FLAG expression supernatants(0.1,1,10 and 100 ng/mL) were used to test the ability of THP-1 cells to migrate through PET membranes.Results Human sFlt-1(1-3) gene was successfully recombined into Ad-sFlt-1/FLAG.sFlt-1(1-3) was expressed in L929 cells transfected with Ad-sFlt-1/FLAG as detected by ELISA and immunofluorescent staining.High levels of sFlt-1/FLAG fusion proteins were present in the culture supernatant of Ad-sFlt-1/FLAG-transfected L929 cells.The amount of sFlt-1/FLAG protein in the culture supernatant was 503.7 ng/mL.At VEGF concentration of 1,10 and 100 ng/mL,more THP-1 cells migrated than the control,the difference was statistically significant(P<0.05).THP-1 cell migration in the presence of 100 ng/mL sFlt1(1-3)/FLAG was about 25.7% of that in the absence of sFlt1(1-3)/FLAG.Conclusion VEGF is able to elicit a migratory response in THP-1 cells,and sFlt-1(1-3) is an effective inhibitor of THP-1 migration towards VEGF.