碱性成纤维细胞生长因子基因转染对鼠胎肝干细胞培养的优化作用

Effect of basic fibroblast growth factor gene transfection on the proliferation of fetal liver stem cells

目的观察以重组腺相关病毒(rAAV)为载体介导碱性成纤维细胞生长因子(bFGF)基因转染对体外培养的鼠胎肝干细胞增殖及细胞周期的影响。方法体外培养鼠胎肝干细胞,经rAAV作为载体介导bFGF基因转染,分为对照组、空载病毒转染组和bFGF转染组。用逆转录PCR和蛋白质印迹法检测鼠胎肝干细胞转染前、后bFGF基因和蛋白的表达。应用细胞生长曲线和CCK-8法观察细胞生长速度;采用流式细胞术测定细胞周期分布的变化。结果转染bFGF后,bFGF转染组与对照组、空载病毒转染组相比,bFGF基因和蛋白水平均有表达,细胞的生长速度增快(q=20.43,P=0.001;q=26.52,P=0.001),G0/G1期细胞减少,S期细胞增多(q=72.56,P=0.000;q=32.28,P=0.001)。结论通过rAAV作为载体介导bFGF基因转染能促进体外培养的鼠胎肝干细胞增殖,对其培养有优化作用。

Objective To investigate the effect of basic fibroblast growth factor （bFGF） gene transfection mediated by recombinant adeno-associated virus （rAAV） as a vector on the proliferation and cell cycle of cultured fetal liver stem cells in vitro. Methods Fetal liver stem cells were cultured by the suspension culture in vitro, and were transfected by bFGF gene mediated by rAAV as a vector. Fetal liver stem cells were divided into 3 groups： control group, airborne virus transfection group, and bFGF group. Reverse transcription-PCR and western blotting were used to assess the expression of bFGF gene and protein on fetal liver stem cells. Cellular proliferation was determined by the cell growth curve and CCK-8 assay. The cell cycle was analyzed by flow cytometry. Results After bFGF transfection, in the bFGF transfection group, the expression of bFGF mRNA and protein content were increased, the cell growth speed was significantly faster （ q = 20.43, P = O. 001 ; q = 26.52, P = O. 001 ） , and the number of C~/G1 phase cells was decreased and cell number in S phase was increased （ q = 72.56, P = 0. 000 ; q = 32.28, P = O. 001 ）, as compared with the control group and airborne virus group. Conclusion The bFGF gene transfection through rAAV as a vector can promote proliferation of fetal liver stem cells cultured and optimization of the culture.