可溶性FasL联合survivin RNA干扰诱导肺腺癌细胞凋亡  

Soluble FasL combined with survivin RNA interference induced apoptosis of lung adenocarcinoma

作　　者：刘黎明[1] 陈昌杰[2] 陈正徐[2] 胡博[2] 段光军[1] 胡华成[1] 

机构地区：[1]苏州大学附属第二医院呼吸内科,苏州215004 [2]蚌埠医学院检验系,蚌埠233004

出　　处：《肿瘤》2007年第8期615-619,共5页Tumor

摘　　要：目的:探讨可溶性FasL(sFasL)联合survivin RNA干扰对肺腺癌细胞A549增殖和凋亡的影响。方法:构建survivin特异RNA小干扰表达载体,DNA测序,转染A549细胞。G418(geneticin)筛选稳定表达survivin RNA小干扰细胞株,荧光实时定量RT-PCR、免疫印迹(Western blot)和免疫组化法检测survivin mRNA和蛋白表达。sFasL作用于转染后细胞,四甲基偶氮唑蓝(MTT)法和流式细胞仪检测细胞活性和凋亡率。结果:成功构建survivin-siRNA表达载体,筛选出稳定表达单克隆细胞。Survivin mRNA和蛋白表达分别下降76.4%(P<0.01)和84.5%,免疫组化也显示survivin下调。sFasL联合survivinRNA干扰细胞,光密度值在各时间点较siRNA组、sFasL组和对照组均明显降低(P<0.01),抑制率增加;凋亡率较siRNA组、sFasL组和对照组明显增加(P<0.01)。结论:Survivin RNA干扰表达载体可降低survivin的表达。sFasL联合survivin RNA干扰较单用sFasL或survivin RNA干扰能更有效地抑制肺腺癌细胞A549增殖,促进凋亡。Objective：To investigate the anti-proliferative and pro-apoptotic effects of soluble Fas ligand （sFasL） combined with survivin RNA interference on A549 lung adenocarcinoma cells in vitro. Methods：The expression vector of survivin small interfering RNA （siRNA） was constructed and confirmed by sequencing. The survivin siRNA expression vector was transfected into A549 cells and the stable cell line expressed survivin small interfering RNA were selected by G418 （geneticin） screening. The mRNA and protein levels of survivin were tested by fluorescent quantitative real-time reverse transcription polymerase chain reaction （ FQ-RT-PCR）, Western blot, and immunohistochemistry. After the transfected cells were treated with sFasL, the cell proliferation and apoptosis were assessed by MTT assay and flow cytometry. Results：Survivin siRNA expression vector was successfully constructed and stable A549 cells were selected. The mRNA and protein levels of survivin decreased by 76.4% （ P 〈0.01 ） and 84.5% , respectively. Immunohistochemistry showed that the survivin was down-regulated. Cells in siRNA ＋ sFasL group had lower proliferation rate at every time point than that of single sFasL group, siRNA group and control group. The difference was significant （P 〈0.01 ）. The apoptotic rate was significantly higher in siRNA ＋ sFasL group than that of sFasL group, siRNA group, and control group [ （ 18.6 ± 2.93 ） % vs （ 10. 12± 2.31 ） %, （ 10.09 ± 1.31 ） %, （ 1.14± 0.21 ） %, P 〈 0.01 ]. Conclusions： Survivin siRNA expression vector significantly inhibites the expre- ssion of survivin in A549 cells. The sFasL combined with survivin siRNA interference can more effectively inhibit the proliferation and induce the apoptosis than single sFasL or survivin RNA interference treatment in lung adenocarcinoma A549 cells

关 键 词：细胞系 肿瘤 RNA干扰 细胞凋亡 Fas配体蛋白 

分 类 号：R730.54[医药卫生—肿瘤]