【摘要】 目的探讨survivin基因表达对人肺纤维母细胞(humanlungfibroblast,HLF)增殖、凋亡的影响。方法构建survivin基因真核表达质粒pcDNA3.1/SVN;利用脂质体转染法将其转入不表达survivinmRNA的HLF细胞系。连续培养10周,筛选出稳定表达的阳性克隆(HLF/SVN)及转空质粒对照(HLF/K)。用RT-PCR、免疫组化、Westernblot检测细胞中survivin、bcl-2和caspase3mRNA表达;通过观察细胞生长曲线、细胞凋亡指数,探讨转染survivin基因对HLF细胞生长、凋亡的影响。结果转染pcDNA3.1/SVN后,在4、6、8和10周HLF/SVN细胞查见survivinmRNA表达;免疫组化和Westernblot见HLF/SVN细胞survivin阳性表达;转染后caspase3mRNA受到抑制,表达减弱或消失。HLF/SVN细胞凋亡指数(0.3%、1.0%、1.5%)皆低于HLF(0.9%、4.3%、2.1%)及HLF/K(0.8%、1.4%、2.8%)。细胞生长曲线示HLF/SVN细胞生长快于HLF和HLF/K,差异有统计学意义(Р﹤0.05)。结论survivin基因能促进HLF细胞生长,可能通过抑制凋亡和促进细胞分裂而发挥作用。更多还原

【Abstract】 [Objective] To study the effect of survivin expression on the amplification and apoptosis of human lung fibroblast(HLF)cells.[Methods] Eukaryotic expression plasmid pcDNA3.1/SVN was constructed and transfected into the HLF cell line without expression of survivin mRNA by lipofection technique.After cultured 10 weeks,the positive cells(HLF/SVN)of transfected plasmid pcDNA3.1/SVN and control cells(HLF/K)of transfected empty plasmid were selected.Expression of survivin,bcl-2 and caspase3 mRNA in the HLF cells were detected by RT-PCR,immunohistochemstry,and Western blot.The effects of survivin mRNA on the amplification and apoptosis of HLF cell were monitored by the growth curve and apoptotic indexes(AI).[Results] After transfecting pcDNA3.1/SVN,the cells of expression survivin mRNA in HLF/SVN were detected at 4 week,6 week,8 week,and 10 week.Expression of survivin protein was detected by immunohistochemstry and Western blot.Expression of caspase3 was reduced or no expression.Apoptosis index was lower in HLF cells of transfected pcDNA3.1/SVN(0.3%,1.0%,1.5%)than untransfection ones(0.9%,4.3%,2.1%)or transfected empty plasmid(0.8%,1.4%,2.8%).The amplification of HLF/SVN was increased than HLF and HLF/K cells by growth curve.There exist a significant difference in HLF/SVN and HLF,HLF/K cells(P﹤0.05).[Conclusion] Survivin gene maybe play an important role in amplification of HLF cells.This change might be attributed to inhibit apoptosis and increas mitosis of cells.更多还原