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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 K562 cells. Methods The cDNA sequence of SHP1 gene was cloned by RT-PCR and cloned into eukaryotic expression vector pcDNA3.0. The gene was overexpressed in K562 cells. The apoptosis of K562 cells was detected by Hoechst33258 staining and FACS (AnnexinⅤ-PI double-labeled) staining. Cell differentiation was analyzed by benzidine blue staining and GPA expression. Results The pcDNA3-SHP1 transfected K562 cells, RT-PCR, Western blot analysis confirmed SHP1 expression in K562 cells. After 48 h of transfection, apoptosis of K562 cells was observed. The apoptosis rate of K562 cells was 16.84% by AnnexinⅤ-PI double-labeled FACS analysis, which was significantly different from that of transfected K562 cells (6.23%) (P = 0.000) . The positive rate of benzidine blue staining cells was 14.67%, the GPA expression rate was 19.38%, which was also significantly different from that of the blank vector transfected group (P = 0.005). Conclusion SHP1 can effectively induce apoptosis and erythroid differentiation in K562 cells.