Chen Y B, Zhang Q X, Butler C E, Ye Y W, Zhang L P, Dong J L, Chen C H, Han Y
Department of Plastic Surgery,University of Texas,MD Anderson Cancer Center,Houston,77030,USA.
Nephrology Division,Department of Medicine,Baylor College of Medicine,Houston,77030,USA.
Lin Chuang Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2016 Jun 20;30(12):966-971. doi: 10.13201/j.issn.1001-1781.2016.12.011.
To explore liposome-mediated transfection of human adipose-derived stem cells (hASCs) with vascular endothelial growth factor(VEGF) gene and to investigate the expression of VEGF after transfection.Lipoaspirate was digested using collagenase.Cell pellet was harvested and subcultured to passage 4.Phenotype was detected with flow cytometry and multilineage differentiation was induced for the identification of hASCs.hASCs was transfected with pIRES2-EGFP-VEGF plasmid using DOTAP liposome.The intracellular expression of VEGF was detected by immunofluorescent staining and the VEGF concentration in supernatant was analyzed by ELISA.1 ml lipoaspirate yielded(4.38±0.21)×10⁵ cells.hASCs on passage 4 showed high expression of CD90(81.49%) and low expression of CD19(6.37%),CD31(14.91%),CD34(17.56%) and CD45(15.39%).GFP and VEGF were observed in transfected hASCs.The transfection efficiency was(43.69±18.53)%.Untransfected hASCs did not express GFP but low level of VEGF.The optical density of VEGF intransfected hASCs is 2.13 fold of untransfected hASCs.The VEGF concentration in supernatant of transfected hASCs significantly increased over time and exhibit statistic differences compared with untransfected hASCs(<0.05).hASCs were successfully transfected with pIRES2-EGFP-VEGF plasmid using DOTAP liposome.The post-transfection expression and secretion of VEGF remarkably increased.
探讨脂质体介导血管内皮生长因子（VEGF）基因转染人脂肪来源干细胞（hASCs）以及转染后VEGF的表达情况。用胶原酶消化抽脂物。收获细胞沉淀并传代培养至第4代。通过流式细胞术检测细胞表型，并诱导多向分化以鉴定hASCs。使用DOTAP脂质体将pIRES2-EGFP-VEGF质粒转染hASCs。通过免疫荧光染色检测VEGF的细胞内表达，并通过ELISA分析上清液中VEGF的浓度。1 ml抽脂物产生（4.38±0.21）×10⁵个细胞。第4代hASCs高表达CD90（81.49%），低表达CD19（6.37%）、CD31（14.91%））、CD34（17.56%）和CD45（15.39%）。在转染的hASCs中观察到绿色荧光蛋白（GFP）和VEGF。转染效率为（43.69±18.53）%。未转染的hASCs不表达GFP，但有低水平的VEGF表达。转染hASCs中VEGF的光密度是未转染hASCs的2.13倍。转染hASCs上清液中VEGF浓度随时间显著增加，与未转染hASCs相比有统计学差异（<0.05）。使用DOTAP脂质体成功将pIRES2-EGFP-VEGF质粒转染hASCs。转染后VEGF的表达和分泌显著增加。
