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目的:研究CCAAT/增强子结合蛋白α(CCAAT/enhancer-binding protein-alpha,C/EBPα)对K562细胞株分化和凋亡的影响及对相关基因的调控,为慢性粒细胞白血病的治疗提供新的治疗靶点。方法:将C/EBPα表达质粒pEGFP-C/EBPα及空载体对照质粒pEGFP分别经阳离子脂质体2000介导转染K562细胞,用G418筛选出C/EBPα稳定表达细胞株。Wright-Giemsa染色观察细胞形态学变化,FCM分析细胞表面分化抗原CD11b的表达、细胞周期及细胞凋亡,电子显微镜观察细胞凋亡,RT-PCR和Western印迹法检测细胞中相关基因Per2、cyclin B1和C-myc的表达。结果:筛选得到稳定表达C/EBPα的细胞株pEGFP-C/EBPα-K562。与空载体转染组及对照组细胞相比,转染组K562细胞分化明显,同时粒系细胞表面分化抗原CD11b表达增加;细胞周期分析发现,转染组细胞中G2期细胞增多,与空载体组和对照组相比,差异有统计学意义(P<0.05),同时出现细胞凋亡峰。细胞凋亡检测结果显示,转染组细胞凋亡明显增加(21.1%),与空载体组(6.0%)和对照组(4.2%)比较,差异有统计学意义(P<0.05);电子显微镜观察发现,转染组细胞中出现染色质浓集、断裂和核固缩等现象,并见凋亡小体;RT-PCR和Western印迹法检测发现,C/EBPα明显上调Per2 mRNA和蛋白表达,抑制cyclinB1、C-myc mRNA和蛋白的表达。结论:C/EBPα能促进K562细胞分化,并诱导细胞凋亡,其机制可能是通过对细胞周期相关基因的调控来实现的。
Objective: To investigate the effects of CCAAT / enhancer-binding protein-alpha (C / EBPα) on the differentiation and apoptosis of K562 cell line and its regulation of related genes, and to provide new treatment for chronic myeloid leukemia Therapeutic targets. Methods: The C / EBPα expression plasmid pEGFP-C / EBPα and empty vector control plasmid pEGFP were transfected into K562 cells with cationic liposome 2000 respectively. The stable C / EBPα-expressing cell line was screened by G418. Wright-Giemsa staining was used to observe the cell morphological changes. The expression of cell surface differentiation antigen CD11b, cell cycle and apoptosis were analyzed by FCM. Cell apoptosis was observed by electron microscopy. Per2 and cyclin B1 were detected by RT-PCR and Western blotting. And C-myc expression. Results: The cell line stably expressing C / EBPα was screened for pEGFP-C / EBPα-K562. Compared with the empty vector transfected group and the control group, the K562 cells in the transfection group were significantly differentiated and the expression of CD11b was increased. The cell cycle analysis showed that the number of G2 phase cells in the transfection group was increased, Compared with the control group, the difference was statistically significant (P <0.05), at the same time, the apoptosis peak appeared. The results of apoptosis showed that the apoptosis of transfected cells was significantly increased (21.1%) compared with the control group (6.0%) and empty vector group (4.2%), the difference was statistically significant (P <0.05) The results of RT-PCR and Western blotting showed that C / EBPα significantly up-regulated the expression of Per2 mRNA and protein, Inhibit cyclinB1, C-myc mRNA and protein expression. Conclusion: C / EBPα can promote the differentiation of K562 cells and induce apoptosis. The mechanism may be through the regulation of cell cycle related genes.