SHP1基因诱导慢性粒细胞白血病K562细胞凋亡及红系分化

SHP1 gene induces apoptosis and erythroid differentiation in human erythromyeloblastoid leukemia cell line K562

目的探讨SHP1基因在诱导K562细胞凋亡及红系分化中的作用。方法应用RT-PCR法克隆SHP1基因全长cDNA序列并克隆于真核表达载体pcDNA3.0,脂质体转染使其基因在K562细胞中过表达。Hoechst33258染色和FACS(AnnexinⅤ-PI双标)分析检测转基因后K562细胞凋亡;联苯胺蓝染色和血型糖蛋白A(GPA)表达检测分析细胞分化情况。结果 pcDNA3-SHP1转染K562细胞,RT-PCR、蛋白免疫印迹分析证实SHP1在K562细胞中表达。转染48h后,K562细胞出现凋亡,AnnexinⅤ-PI双标FACS分析细胞凋亡率为16.84%,与转染空载体pcDNA3.0(6.23%)相比差异有统计学意义(P=0.000)。联苯胺蓝染色细胞阳性率14.67%,GPA表达率19.38%,与转染空载体组比较差异也有统计学意义(P=0.005)。结论 SHP1能有效地诱导K562细胞凋亡与红系分化。

Objective To investigate the role of SHP1 gene in inducing apoptosis and erythroid differentiation in human erythromyeloblastoid leukemia cell line K562.Methods The full length cDNA of SHP1 gene was cloned by RT-PCR and was subcloned into mammalian expression vector pcDNA3.0.The cDNA sequence of the cloned gene was validated by enzyme digestion and DNA sequencing.Then the recombinant plasmid was used to transfect K562 cells via lipofectin.The apoptosis of K562 cells was examined by Hoechst 33258 staining assay and Annexin Ⅴ/PI double-labeled assay;the differentiation of K562 cells was observed by benzidine staining and expression of glycophorin A （GPA）.Results RT-PCR and Western blotting analysis showed expression of SHP1 in K562 cells after transfection with pcDAN3-SHP1 plasmid.Apoptotic cells were detected in the K562 cells 48 h after treatment with pcDNA3-SHP1,with the apoptosis rate being 16.84%,which was significantly higher than that in cells transfected with pcDNA3.0 （6.23%,P=0.000）.The positive rate of benzidine staining was 14.67% and the positive rate of GPA expression was 19.38% in cells treated with pcNDA3-SHP1,both were significantly different from those in the cells transfected with pcDNA3.0 （P=0.005）.Conclusion Over-expression of SHP1 can effectively induce apoptosis and erythroid differentiation in K562 cells.