腺病毒介导的KGF基因修饰促进表皮干细胞增殖

Effect of recombinant adenovirus vector carrying human KGF gene on the proliferation of human epidermal stem cells

目的构建KGF腺病毒表达载体并转染表皮干细胞,观察其对表皮干细胞增殖的影响。方法 RT-PCR自人成纤维细胞中扩增KGF基因片段,pAD-easy I腺病毒系统构建KGF腺病毒表达载体,以MOI=50感染表皮干细胞;MTT法检测表皮干细胞增殖,流式分析细胞周期变化。结果 RT-PCR成功扩增KGF基因片段,成功构建的KGF腺病毒质粒经Pac I内切酶酶切获得4.5 kbp的特异条带,MOI=50的病毒转染效率超过90%;KGF基因修饰后的表皮干细胞增殖效率与对照组比较有显著差异(P<0.05),并且G2/M期细胞数量明显增多。结论成功构建腺病毒KGF表达载体,腺病毒载体介导的KGF基因修饰促进了表皮干细胞的增殖。

Objective To construct a recombinant adenovirus vector carrying human keratinocyte growth factor（KGF） gene,and investigate its proliferative effect on the transferred primary human epidermal stem cells.Methods RT-PCR was used to amplify the sepuence of human keratinocyte growth factor.Recombinant adenoviral vector containing the sequence was prepared using pAD-easy-I system.The primary human epidermal stem cells were transfected with the recombinant virus vector.Under the fluorescence microscope,the transfection efficiency was determined.MTT assay was used to analyze the proliferation of hESCs.Cell cycle of hESCs was evaluated by flow cytometry.Results KGF gene was amplified as expected.The recombinant adenoviral plasmid could be digested with Pac I to produce a fragment of 4.5kbp.The transfection efficiency of the recombinant virus at the MOI of 50 was 90%.The proliferation of transgenic cells significantly increased compared to that of controls,evaluated by MTT and the ratio of G2/M cell of the transgenic group.Conclusions The recombinant adenovirus vector containing KGF gene was successfully constructed.The KGF transgenic hESCs showed favourable proliferative potential.