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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 investigate the effect of survivin gene expression on the proliferation and apoptosis of human lung fibroblasts (HLF). Methods The survivin gene eukaryotic expression plasmid pcDNA3.1 / SVN was constructed and transfected into HLF cell line without survivin mRNA by lipofection. After continuous culture for 10 weeks, stable positive clones (HLF / SVN) and empty plasmid control (HLF / K) were screened out. The expressions of survivin, bcl-2 and caspase3 mRNA were detected by RT-PCR, immunohistochemistry and Western blot. The cell growth curve and apoptosis index were observed to investigate the effect of survivin gene on the growth and apoptosis of HLF cells. Results The expression of survivin mRNA was detected in HLF / SVN cells at 4, 6, 8, and 10 weeks after transfection with pcDNA3.1 / SVN. The expression of survivin in HLF / SVN cells was detected by immunohistochemistry and Western blot, and the expression of caspase3 mRNA was inhibited after transfection Weaken or disappear HLF / SVN apoptosis index (0.3%, 1.0%, 1.5%) were lower than HLF (0.9%, 4.3%, 2.1%) and HLF / K (0.8%, 1.4%, 2.8%). Cell growth curve showed that HLF / SVN cells grew faster than HLF and HLF / K, the difference was statistically significant (Р <0.05). Conclusion The survivin gene can promote the growth of HLF cells and may play a role by inhibiting apoptosis and promoting cell division.