转染survivin对HLF细胞增殖及凋亡的影响

Effect of Transfected Survivin on Amplification and Apoptosis of HLF

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。

[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 ceils （HLF/SVN） of transfected plasmid poDNA3.1/SVN and control cells（HLF/K） of transfected empty plasmid were selected. Expression of survivin, bcl-2 and caspase3 mRNA in the HLF ceils 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 poDNA3.1/ 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 ceils of transfected poDNA3.1/SVN （0.3%, 1.0%, 1.5%）than untransfection ones （0.9%,4.3%,2.1%）or transfectod empty plasmid （0.8%,1.4%,2.8%）. The amplification of HLF/SVN was increased than HLF and HLF/K ceils 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 ceils. This change might be attributed to inh/bit apoptesis and increas mitosis of ceils.