胰岛素样生长因子-1基因转染对人尿源干细胞生物活性的影响

Effects of IGF-1 genetic modification on the bioactivity of human urine-derived stem cells

目的分离、培养并鉴定尿源干细胞(USCs),评估慢病毒载体介导胰岛素样生长因子1(IGF-1)基因感染USCs的安全性及有效性,探究IGF-1对USCs增殖、迁移能力的影响,为后续USCs-基因联合移植治疗勃起功能障碍(ED)动物模型提供可靠的体外实验证据。方法采用差速贴壁法分离培养原代USCs并鉴定。慢病毒载体介导IGF-1转染USCs,采用Western blot和PCR检测IGF-1表达情况,采用CCK-8实验、Transwell小室分析IGF-1对USCs的增殖及迁移能力影响。结果成功分离人USCs并传代培养。成骨、成脂分化后,茜素红S、油红O染色阳性;流式细胞技术检测结果显示,USCs表达间充质干细胞标志物CD44、CD73、CD90、CD146,不表达造血干细胞标志物CD31、CD45。重组USCs能稳定持续高表达IGF-1蛋白;重组USCs增殖、迁移能力显著高于对照组USCs(P<0.05),而转染慢病毒空载体USCs与对照组USCs比较,差异无统计学意义(P>0.05)。结论USCs取材简便且无创,分化和增殖能力强;慢病毒载体介导IGF-1转染USCs安全有效,IGF-1能够促进USCs增殖、迁移,为后续USCs-基因联合移植治疗ED奠定基础。

Objective To isolation,culture and identification of urine-derived stem cells(USCs),evalu-ate the safety and effectiveness of lentiviral vector-mediated insulin-like growth factor 1(IGF-1)gene infection of USCs,and explore the effects of IGF-1 on proliferation and migration of USCs,provide reliable in vitro experimental evidence for the subsequent usC USCs-gene combination transplantation in the treatment of erectile dysfunction(ED)animal model.Methods The primary USCs was isolated and cultured by differential adhesion method and i-dentified.Lentiviral vector mediated IGF-1 transfected USCs,and the expression of IGF-1 was detected by Western blot and PCR.CCK-8 assay and Transwell chamber were used to analyze the effects of IGF-1 on the proliferation and migration of USCs.Results USCs were successfully isolated and sub cultured.The induced differentiation experi-ments identified multipotential differentiation ability of USCs.Flow cytometry showed that USCs expressed mesen-chymal stem cell markers CD44,CD73,CD90 and CD146,but did not express hematopoietic stem cell markers CD31 and CD45.The recombinant USCs can stably overexpress IGF-1.The proliferation and migration of recombi-nant USCs were significantly higher than those of the control group.Conclusions Urine-derived stem cells are non-invasive and convenient to be obtained.They have strong differentiation and proliferation ability.Transfection of USCs with IGF-1 mediated by lentivirus vector is safe and effective.IGF-1 can promote proliferation and migration of USCs,laying a foundation for subsequent USCS-gene combined transplantation in the treatment of ED.