SOCS-3过表达对红系基因表达的影响

目的:建立能稳定、高效表达细胞因子信号转导抑制因子-3(SOCS-3)的细胞株SOCS-3-K562,为探讨SOCS-3在造血发育中的作用奠定基础。方法:通过重组慢病毒系统感染人红白血病细胞K562,采用流式细胞分选术,根据绿色荧光蛋白表达情况,获得稳定高表达SOCS-3的K562细胞;利用实时荧光定量PCR和蛋白质印迹实验,比较分选获得的细胞与对照细胞的SOCS-3表达差异;利用半定量PCR检测SOCS-3表达升高对K562红系发育相关基因GATA-1、β-globin表达水平的影响。结果:构建了人SOCS-3慢病毒表达载体;与对照组相比,通过流式细胞分选获得的K562细胞的SOCS-3基因表达水平升高8.05倍,蛋白表达水平升高3倍;SOCS-3表达升高后,K562细胞的GATA-1、β-globin基因表达受到明显抑制。结论:SOCS-3在造血发育中有重要的调控作用,而对其表达进行改变将在规模化的造血细胞定向诱导研究中发挥重要作用。

Overexpression of SOCS-3 Decrease Expression of Erythriod Genes

Objective： To establish K562 cell line expressing suppressor of cytokines signals 3（SOCS-3） stably and build foundation to study the regulation mechanism of SOCS-3 for hematopoietic development. Methods： The Expression vectors of SOCS-3 were constructed and transferred into the K562 cell lines stably by lentiviral system. The efficiency of virus transfection was identified by expression of GFP observed 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 SOCS-3 expression was detected by real-time PCR and Western blotting. The expression of erythropoietic gene β-globin and GATA-1 in K562 cells was examined by RT-PCR. Results： K562 cell line expressing SOCS-3 have been established. Compared with the control K562 cells, SOCS-3 gene expression of GFP＋K562 ceils increased 8.05 fold at mRNA level and increased about 3 fold at the protein level. Gene expression of β-globin and GATA-1 were suppressed with SOCS-3 gene overexpression. Conclusion： SOCS- 3 played an important role in hematopoietic development and it may be a useful new way for production of erythroid lineage cells in large scale by altering the expression of SOCS-3.