siRNA沉默HIF-1α对胎肝基质细胞SDF-1α表达的影响  

Effect of Silencing Hypoxia Inducible Factor 1α by siRNA on The Expression of Stromal Cell Derived Factor 1α in Fetal Liver Stroma Cell

作　　者：吉蕾[1] 习佳飞[1] 袁红丰[1] 张鹏[1] 刘雨潇[1] 王韫芳[1] 施双双[1] 陈琳[1] 南雪[1] 白慈贤[1] 裴雪涛[1] 

机构地区：[1]军事医学科学院输血医学研究所干细胞与再生医学研究室,北京100850

出　　处：《生物化学与生物物理进展》2007年第10期1040-1048,共9页Progress In Biochemistry and Biophysics

基　　金：国家高技术发展计划重大专项(2006AA02A107);国家重大基础研究发展计划(973)(2005CB522702);北京市科委重大项目培育专项(Z0005190043331)资助项目.~~

摘　　要：为了研究低氧诱导因子(hypoxia inducible factor-1alpha,HIF-1α)在干细胞增殖分化过程中的作用机制,构建了2个HIF-1α慢病毒siRNA干涉载体,并转染人胎儿肝脏基质细胞(fetal liver stromal cell,FLSC).根据绿色荧光蛋白的表达评估转染效率后进行流式细胞分选,获得高表达慢病毒干涉载体的细胞.实时荧光定量PCR和蛋白质印迹检测了转染细胞中HIF-1α基因的干涉效率,结果显示,与对照组相比,常氧下培养的细胞HIF-1α基因表达量仅为其相对表达量的18.8%和25.5%,干涉效率分别为81.2%和74.5%,低氧处理后的细胞HIF-1α相对表达量分别为对照组的21.2%和29.3%,干涉效率分别为78.8%和70.7%,均具有显著差异.蛋白质印迹结果显示,在蛋白质水平表达也明显受抑制,且重组干涉质粒pSicoR-HIF-1α1的干涉效应较强.RT-PCR、免疫荧光和ELISA法检测了沉默HIF-1α后胎肝基质细胞衍生因子1α(SDF-1α)在RNA和蛋白质水平的表达变化,干涉后细胞SDF-1α的表达明显减低.低氧条件下SDF-1α基因的调控作用有可能是通过低氧激活HIF-1α而诱导产生的,HIF-1α在干细胞增殖分化的分子调控机制中具重要作用.Hypoxia-inducible factor-1α （HIF-1α is a key regulator of the physiological response to hypoxia. To study the regulation mechanism of HIF-1α in proliferation and differentiation of stem cells, two small interference RNA expression vectors of HIF-1α were constructed, and transfected into the fetal liver stromal cell lines （FLSCs） stably by lentiviral system. The efficiency of virus transfection was identified by expression of green fluorescence protein （GFP） analyzed by fluorescence microscope, then the high GFP expression FLSCs were sorted by fluorescence-activated cell sorting （FACS） according to strong GFP expression. Analysis of efficiency of RNA interfering on HIF-1α was detected by real time-PCR and Western-blot. The HIF-1α gene expression at mRNA level of FLSCs transfected by lentivirus plasmids pSicoR-HIF- 1 α- 1 and pSicoR-HIF- 1α-2 are 18.8% and 25.5% of the FLSCs transfected by the control lentivirus under 20% 02; 21.2% and 29.3% under hypoxia. The HIF-The protein were down regulated by siRNA. The pSicoR-HIF 1 showed higher interfering efficiency than pSicoR-HIF2. The expression of SDF-1α gene in HIF-1α silencing FLSCs were detected by RT-PCR, fluorescent immunocytochemistry analysis and ELISA. The mRNA and protein of SDF-1α gene were down regulated after silencing of HIF-1α. Therefore, the regulation of SDF-1α gene under hypoxia could be activated by HIF-1α, that plays an important role in the influence on the proliferation and differentiation of stem cell.

关 键 词：siRNA 基因沉默 低氧诱导因子1Α 慢病毒 基质细胞 基质细胞衍生因子1Α 

分 类 号：Q78[生物学—分子生物学] Q813.1