脂质体介导人骨形态发生蛋白2基因转染对兔脂肪干细胞生长增殖的影响

Effects of liposome-mediated human bone morphogenetic protein-2 gene transfection on growth and proliferation of rabbit adipose-derived stem cells

背景:脂质体对细胞具有毒副作用,而外源性基因的胞内导入亦在细胞的代谢方面有所影响,因此脂质体介导人骨形态发生蛋白2基因转染脂肪干细胞对其在生长、增殖方面有无显著影响值得探讨。目的:观察脂质体介导的人骨形态发生蛋白2基因转染对兔脂肪干细胞生长、增殖的影响。方法:从兔脂肪组织中提取脂肪干细胞,体外培养传代,取第4代细胞分别转染人骨形态发生蛋白2基因和增强型绿色荧光蛋白基因,倒置显微镜下连续观察转染后的细胞形态变化及生长情况。荧光显微镜下计数转染后48h可发绿色荧光细胞的个数估算瞬时转染效率,MTT法绘制染后细胞的生长曲线,并计算群体倍增时间。结果与结论:转染后48h,细胞体积变大,胞浆丰富,部分呈圆形或椭圆形改变。荧光显微镜下计数瞬时转染率为(18.0±0.42)%。MTT法绘制细胞生长曲线发现:转染后细胞较未转染的细胞生长速率略慢,但转染与未转染组细胞的生长曲线大致一样,均呈"S"形,且两者的总体趋势变化不大。转染后人骨形态发生蛋白2组的细胞群体倍增时间为55.51h,EGFP组群体倍增时间约为53.58h,未转染组的群体倍增时间46.10h,差异无显著意义(P>0.05)。提示脂质体可以介导人骨形态发生蛋白2基因和绿色荧光蛋白基因转染兔脂肪干细胞,并对细胞的生长、增殖无明显影响。

BACKGROUND：Liposome has toxic side effect on cells,but introduction of exogenous genes into cells affects cell metabolism.Therefore,it is worthy to explore effects of liposome-mediated human bone morphogenetic protein-2 （hBMP-2） gene-transfected adipose-derived stem cells （ADSCs） on cell growth and proliferation.OBJECTIVE：To investigate the effects of liposome-mediated hBMP-2 gene transfection on growth and proliferation of rabbit ADSCs.METHODS：The rabbit ADSCs were extracted from the adipose tissue and subcultured in vitro.The fourth passage of ADSCs were collected and transfected by hBMP-2 gene and enhanced green fluorescent protein （EGFP） gene respectively.The cell morphology and their related growth pattern were continuously observed under the inverted microscope after transfection.At 48 hours after transfection,the transient transfection efficiency was measured by fluorescence microscopic counting.MTT assay was used to drawn the cell growth curve and to calculate the colony doubling time.RESULTS AND CONCLUSION：At 48 hours after transfection,cell volume became large,with abundant cytoplasm,and some were round or oval.The transient transfection rate was about （18.0±0.42）% under fluorescence microscope.According to the cell growth curves drawn by MTT assay,the transfected cells grow at a slower rate compared with the untransfected cells,but both of them roughly had the same growth curve,like the shape of ＂S＂,and little change in their overall trends.After transfection,the population doubling time was 55.51 hours in the hBMP-2 group,53.58 hours in the EGFP group and 46.10 hours in the non-transfected group.There was no significant statistical difference （P 0.05）.Results have indicated that liposomes can mediate the hBMP-2 gene and EGFP gene transfected rabbit ADSCs and it has no effect on their growth and proliferation.