NF-H基因参与K562/A02细胞多药耐药机制形成的研究

Identification of differentially expressed genes involved in multidrug resistance in K562/A02 by cDNA microarray

目的 分析K5 6 2 /A0 2细胞多药耐药性 (MDR)分子机制 ,寻找可能参与K5 6 2 /A0 2细胞耐药机制的新基因。方法 通过长期逐步增加K5 6 2细胞培养液中阿霉素 (ADM)的浓度 ,诱导出多药耐药细胞株K5 6 2 /A0 2 ,利用cDNAmicroarray比较K5 6 2和K5 6 2 /A0 2基因表达的差别 ,从中选择NF H基因进行RT PCR和免疫细胞化学验证 ,并利用反义核酸技术和细胞内ADM浓度的测定 ,进一步验证该基因与K5 6 2 /A0 2细胞多药耐药的关系。结果 通过比较发现 ,有 12个基因可能参与了K5 6 2 /A0 2细胞的耐药机制 ,其中 7个基因在K5 6 2 /A0 2中被下调 ,5个基因被上调。本研究结果显示 ,NF H基因在K5 6 2 /A0 2细胞中高表达 ,并且 ,将NF H和mdr1反义核酸同时转入K5 6 2 /A0 2细胞后 ,可明显提高细胞内ADM浓度 ,而单独转入NF H反义核酸效果不明显。结论 K5 6 2 /A0 2细胞耐药表型的形成是多因素的 ,除了P 糖蛋白 (P gp)等常见因素外 ,可能还有NF

Objective To study the molecular mechanism underlying multidrug resistance (MDR) and identify unknown genes that might be involved in drug resistance development in K562/A02 cells. Methods K562/A02 was induced by gradually increasing the ADM concentration in culture medium of K562 cells, the differential expression of associated genes between K562 and K562/A02 was determined with cDNA microarray. Overexpression of neurofilament protein NF H gene in K562/A02 cells was confirmed with RT PCR and immunocytochemistry. Anti sense oligodeoxynucleotides were transfected into K562/A02 cells by lipofectamine in order to further analyze the role of NF H in drug resistance. Results Comparing with the expression profiles, we found upregulation of 5 transcripts and downregulation of 7 transcripts in response to MDR of K562/A02 cells. The overexpression of NF H, one of the 5 upregulated genes, was confirmed. After being treated with antisense oligodeoxynucleotides of NF H and mdr1, the cellular adriamycin concentration increased significantly, but antisense NF H alone did not have significant effect on drug resistance phenotype. Conclusion The development of MDR in K562/A02 cells is multifactorial. NF H may be involved in the drug resistance of K562/A02, which may provide a new marker of diagnosis and a new target of therapy.