慢病毒介导酸性成纤维细胞生长因子基因对脂肪干细胞增殖及周期的影响

摘要

目的 探讨慢病毒介导酸性成纤维细胞生长因子1 (FGF1)基因转染脂肪干细胞(ADSC)对ADSC增殖及周期的影响.方法 取健康脂肪抽吸者脂肪混悬液分离提取ADSC,做ADSC培养、鉴定、成骨细胞诱导分化.将含有目的基因载体质粒pWPXLd-FGF1与辅助质粒psPAX2,pMD2.G共转染293T细胞,获得编码FGF1慢病毒,使用慢病毒转染体外培养的ADSC,荧光显微镜下观察细胞表达绿色荧光蛋白情况,并用蛋白质印迹法检测FGF1在ADSC表达.流式细胞仪检测转染FGF1后ADSC周期情况,EDU法检测转染FGF1后ADSC增殖情况.结果 获得编码FGF1慢病毒.编码FGF1慢病毒感染ADSC后,约70％ ADSC出现绿色荧光,蛋白质印迹法显示编码FGF1慢病毒感染ADSC后FGF1蛋白高表达.与对照组相比,FGF1高表达后ADSC的G2/M期细胞数增加48％,细胞增殖增加75％ (P＜0.05).结论 FGF1基因可促使ADSC进入G2/M期增多,促进ADSC增殖.%Objective To study the effect of the lentivirus encoding acidic fibroblast growth factor transfecting human adipose-derived stem cells (ADSCs) on the cell cycle and proliferation of ADSCs.Methods ADSCs were isolated and extracted by enzymatic digestion from the liposuction aspirate.ADSCs were cultured,identified and osteogenic induced reagent was used to induce differentiation of ADSCs towards bone cells.To obtain lentivirus encoding FGF-1,the plasmid PWPXLd FGF-1 was co-transfected with plasmid psPAX2,pMD2.G in 293T cells.ADSCs were infected with lentivirus encoding FGF-1.Expression of green fluorescent protein (GFP) in infected FGF-1 was observed by fluorescence microscope and expression of FGF-1 in ADSCs was verified by Western blot analysis.Flow cytometry was used to detect the cell cycle of ADSCs infected with lentivirus encoding FGF-1.EDU assay was performed to examine cell viability.Results Lentivirus encoding FGF-1 was obtained.After ADSCs being infected green fluorescence was found in about 70％ ADSCs,and overexpression of FGF-1 protein was detected in infceted cells by Western blot analysis.The percentage of G2/M phase cells was significantly increased compared with the control group,and the proliferation of ADSCs infected with lentivirus encoding FGF-1 was promoted as compared with the control group.Conclusions FGF-1 can enhance G2/M phase of ADSCs and promote the proliferation of ADSCs.