siRNA沉默socs3对红系发育的影响

为了研究细胞因子信号转导分子3(suppressor of cytokine signals-3,SOCS-3)对造血发育的影响,构建了SOCS-3慢病毒siRNA干涉载体,并转染人红白血病细胞株K562.根据绿色荧光蛋白的表达进行流式分选后,获得了高表达慢病毒干涉载体的细胞.实时荧光定量PCR和Western-blot检测了转染细胞中SOCS-3基因的干涉效率,结果显示,与对照组相比,siRNA干涉后K562细胞SOCS-3基因的表达量仅为其相对表达量的22.1%,干涉效率77.9%;Western-blot结果显示,SOCS-3在蛋白质水平表达也明显受抑制.进一步对SOCS-3基因沉默后的K562细胞进行了诱导分化,并采用联苯胺染色法检测K562细胞向红系分化比例变化,免疫荧光染色检测细胞表面抗原的变化,RT-PCR检测造血相关基因的变化.结果发现,SOCS-3沉默后K562细胞向红系的发育能力显著提高.研究结果证明,SOCS-3在造血发育中有重要调控作用,而对其表达进行干涉或沉默将在规模化的红细胞诱导研究中发挥重要作用.

Effect of Silencing SOCS-3 by siRNA on The Erythropoietic Development

To study the regulation mechanism of SOCS-3 (Suppressor of cytokines signals-3) for erythropoietic development, small interference RNA expression vectors of SOCS-3 were constructed and transferred in to the K562 cell lines 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 K562 cells were sorted by fluorescence-activated cell sorting (FACS) according to strong GFP expression. The efficiency of RNA interferencing on SOCS-3 were detected by Real time-PCR and Western blot. The SOCS-3 gene expression at mRNA level of K562 cells transfected by lentivirus plasmids was 22.1% of the K562 cells transfected by the control lentivirus. The SOCS-3 protein expression of K562 cells transfected by lentivirus plasmids was also down regulated. Furthermore, K562 cells transfected by lentivirus plasmids were induced into the erythropoietic cells and the erythropoietic differentiation of K562 cells were examined by benzidine staining, immunocytochemistry and RT-PCR. Then it was found that the K562 cells could be induced into erythroid lineage cells more easily after silencing of SOCS-3. The experiment confirmed the important role of SOCS-3 in erythropoietic development and provided a useful new way for production of erythroid lineage cells.