DOTAP脂质体介导VEGF基因转染人脂肪干细胞及目的基因的表达  

DOTAP liposome-mediated transfection of human adipose-derived stem cells with pIRES2-EGFP-VEGF plasmid and target gene expression

作　　者：陈犹白[1,2] 张启旭[1] Butler C E 吴叶文 张丽萍[3] 董江陵[3] 陈聪慧 韩岩[2] 

机构地区：[1]美国德克萨斯大学MD安德森肿瘤中心整形外科,休斯敦77030 [2]解放军总医院整形修复科,北京100853 [3]美国贝勒医学院医学系肾脏学部 [4]北京美莱美容整形医院口腔美容中心

出　　处：《临床耳鼻咽喉头颈外科杂志》2016年第12期966-971,共6页Journal of Clinical Otorhinolaryngology Head And Neck Surgery

基　　金：国家自然科学基金面上项目(No:81272123;No:81571864)

摘　　要：目的：研究脂质体介导的血管内皮生长因子（VEGF）基因转染人脂肪干细胞（hASCs）及VEGF的表达。方法：胶原酶消化吸脂术脂肪,收获细胞沉淀,培养至第4代时利用流式细胞术检测细胞表型,并诱导其成脂成骨分化,鉴定为hASCs。转染组利用DOTAP脂质体将pIRES2-EGFP-VEGF质粒转入hASCs,对照组为脂质体空载。免疫荧光染色检测hASCs内VEGF的表达,ELISA检测上清液中VEGF浓度的变化。结果：1ml吸脂术脂肪可得到（4.38±0.21）×10^5个细胞,第4代时hASCs高表达CD90（81.49%）,低表达CD19（6.37%）、CD31（14.91%）、CD34（17.56%）和CD45（15.39%）,诱导分化后油红O和茜素红染色均呈阳性。分光光度计显示质粒DNA浓度为595ng/μl。转染组hASCs表达GFP和VEGF,转染效率为（43.69±18.53）%;对照组不表达GFP,但表达较低的VEGF。转染组VEGF的光密度是对照组的2.13倍,其上清液中的VEGF浓度随时间显著上升,与对照组相比差异有统计学意义（P〈0.05）。结论：DOTAP脂质体可成功将pIRES2-EGFP-VEGF质粒转入hASCs,转染后的VEGF表达和分泌水平显著提高。Objective：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.Method：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.Result：1ml lipoaspirate yielded（4.38±0.21）×10^5 cells.hASCs on passage 4showed 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（P〈0.05）.Conclusion：hASCs were successfully transfected with pIRES2-EGFP-VEGF plasmid using DOTAP liposome.The post-transfection expression and secretion of VEGF remarkably increased.

关 键 词：吸脂术 脂肪干细胞 血管内皮生长因子 转染 脂质体 

分 类 号：R349.83[医药卫生—基础医学]