NME3蛋白在急性髓系白血病细胞株HL-60和患者骨髓表达趋势研究

摘要

为了验证HL-60细胞向粒、单两系分化前后非转移细胞蛋白3(NME3)的表达差异,并探讨该蛋白对急性髓系白血病的诊断价值,采用全反式维甲酸(ATRA)和甾体类新药NSC67657分别诱导急性髓系白血病HL-60细胞向粒系和单核系分化,通过细胞化学染色判断细胞分化方向,通过细胞表面分化抗原(CD11b/CD14)检测判断细胞分化程度,通过磷脂酰丝氨酸外翻排除细胞凋亡,建立细胞分化模型,应用RT-PCR和Western blot方法验证NME3基因和蛋白在细胞分化前后的差异表达情况.收集26例临床确诊的各种类型髓系白血病患者及5例正常人骨髓样本,比较分析NME3蛋白在不同样本组中的表达趋势.结果表明:ATRA(2 μmol/L,5d)和NSC67657(10μmol/L,5d)可分别诱导HL-60细胞向粒系和单核系分化,其分化程度达到90％以上且不伴随凋亡发生,NME3基因与蛋白表达在细胞两系分化后明显下降.对患者骨髓有核细胞分析发现,NME3蛋白在急性髓系白血病患者骨髓样本中表达较慢性髓系白血病患者组及正常对照组明显升高.结论:NME3蛋白在HL-60细胞分化后表达下调,与其在患者样本中的表达趋势相符,提示该蛋白可能作为急性髓系白血病潜在的分子标志物.%To verify the differential expression of non-metastasis cell 3 (NME3) protein in HL-60 cells when they were induced to differentiate into monocyte and granulocyte like cells, and study its value in diagnosis of acute myeloid leukemia, all-trans retinoic acid( ATRA)and a new steroidal drug NSC67657 were employed to induce acute myeloid leukemia HL-60 cells into monocyte and granulocyte like cells. Then the cell differentiating direction was observed by chemical staining, the degree of differentiation was determined by surface antigen CD11b/CD14 detection, and the apoptosis was excluded by phosphatidylserine valgus analysis, by which cellular differentiating model was constructed. Furthermore, RT-PCR and Western blot were employed to verify the differentially expression of NME3 before and after differentiation of HL-60 cells. At last, samples from bone marrow nucleated cells of 26 patients with myeloid leukemia, which were diagnosed definitely by clinical doctors, and 5 normal people were chosen. Then the expressing trend of NME3 protein in these testing groups was analyzed by means of comparison. The results showed that ATRA(2 μmol/L for 5 d) and NSC67657( 10 μmol/L for 5 d) could induce HL-60 cells to defferentiate into monocyte and granulocyte like cells above 90% without cell apoptosis. The expression of NME3 gene and protein were down-regulated by the inducers, which was accorded with the screening results that was got using proteomics technology in the former research. The expression of NME3 protein in bone marrow from acute myeloid leukemia patients was elevated significantly as compared to normal persons. It is concluded that the expression level of NME3 protein is down-regulated after cellular differentiation, according with the changing trend in leukemia patients, which imply that NME3 protein may be a potential biomarder for diagnosis of acute myeloid leukemia.