骨髓间充质干细胞作为携带PEDF基因载体的可行性研究

Study to evaluate the feasibility of mesenchymal stem cells as delivery vehicles of PEDFgene

目的探讨采用重组色素上皮源性因子腺病毒(Ad-PEDF)转染小鼠骨髓间充质干细胞(MSCs)后,评估MSCs作为携带PEDF基因载体的可行性。方法 Ad-PEDF在HEK293细胞中扩增、并纯化后。将其转染入MSCs(MSCs-PEDF),用Western Blot和ELISA检测培养上清液中PEDF的表达。通过人脐静脉内皮细胞(HUVECs)成管抑制实验和迁移抑制实验来评估MSCs-PEDF表达的PEDF是否具有生理活性。结果用Ad-PEDF或Ad-LacZ转染MSCs 48小时后,用Western Blot检测细胞培养上清中的PEDF为阳性表达。ELISA检测培养上清中PEDF浓度为(78.8±4.8)ng/ml。HUVECs成管抑制实验和迁移抑制实验显示,MSCs-PEDF的培养液上清可显著抑制HUVECs形成小管及向生长因子迁移。结论 MSCs可以作为使用PEDF基因治疗肿瘤的载体。

Objective In this study,we investigated the effect and mechanism of Ad-PEDF transduced MSCs and evaluated the feasibility of MSCs loaded anti-cancer gene.Methods The adenoviruses encoding PEDF（Ad-PEDF）were amplified in HEK293 cells and purified using the ViraTrapTM Adenovirus Purification Maxiprep Kit.MSCs were infected with Ad-PEDF and the expression of PEDF was determined by Western Blot and enzyme linked immunosorbent assay（ELISA）.The biological activity of PEDF was evaluated by HUVECs tube formation inhibition assay and migration inhibition assay.Results 48 hours after Ad-PEDF or Ad-LacZ transduced,the expression of human PEDF were comfirmed by Western blot analysis and ELISA.HUVECs tube formation inhibition assay and migration inhibition assay showed that the conditioned media from MSCs-PEDF dramatically blocked the tube formation and HUVECs migration.Conclusion Mesenchymal stem cells have the potential application as delivery vehicles of PEDF gene for cancer therapy.