腺病毒介导S24F基因转染人脐静脉内皮细胞对调节活化正常T细胞表达和分泌因子趋化功能的影响

Effects of chemoattractant function of regulated upon activation normal T cell expressed and secreted with adenovirus-mediated gene transfer of S24F of the human umbilical vein endothelial cells

目的 观察腺病毒介导S24F基因转染人脐静脉内皮细胞（HUVECs）对调节活化正常T细胞表达和分泌因子（RANTES）趋化作用的影响.方法 构建pAV-MCMV/S24F-GFP-3 FLAG（Ad-S24F）腺病毒载体,将不含目的基因的病毒载体pAV-MCMV-GFP （Ad-Null）为阴性对照组.分离培养HUVECs后分别转染Ad-S24F和Ad-Null,另设不含腺病毒培养基为空白对照.转染后采用荧光显微镜及Western blot检测重组蛋白表达.采用Transwell小室法分析S24F对RANTES趋化作用的影响.结果 Ad-S24F及Ad-Null构建成功,转染HUVECs后荧光显微镜及Western blot能检测到重组蛋白表达,Ad-S24F转染后能够抑制RANTES诱导的外周血单个核细胞（PBMCs）穿透内皮[Ad-S24F：（9.20 ±0.02）％;Ad-Null：（17.70±0.02）％;空白对照组（15.10±0.01）％]的趋化作用.结论 腺病毒介导S24F基因转染HUVECs能够抑制RANTES的趋化作用.

Objective To investigate the effects of chemoattractant function of regulated upon activation normal T cell expressed and secreted （RANTES） with adenovirus-mediated gene transfer of S24F of the human umbilical vein endothelial cells （HUVECs）.Methods Construction of the recombinant Adenoviruses Vector pAV-MCMV/S24F-GFP-3FLAG （Ad-S24F）,an adenoviral vector containing no transgene pAV-MCMV-GFP （Ad-Null） was used as a control.HUVECs were isolated and cultured in vitro,and were transfected with Ad-S24F （Ad-S24F group）,Ad-Null （Ad-Null group）,or transfected with no virus （control group）.The expression of reconstructive protein after transfection was detected by using Western blotting and fluorescence microscopy in each group.The in vitro transendothelialchemotaxis assay was used to compare RANTES-induced transmigration of peripheral blood mononuclear cells （PBMCs） across HUVECs cultured on the upper Transwell chamber.Results The successful construction of recombinant adenoviral vector carrying S24F gene.RANTES-induced PBMC transendothelialchemotaxis is inhibited by S24F [Ad-S24F：（9.20 ± 0.02） ％ ; Ad-Null：（17.70 ± 0.02） ％ ; control：（15.10 ± 0.01） ％].Conclusion Adenovirus-mediated gene transfer of S24F of the human umbilical vein endothelial cells may inhibit RANTES-induced PBMC transendothelialchemotaxis.