醛脱氢酶基因转导介导的K562细胞对环磷酰胺的耐受性

摘要

本研究的目的是观察经醛脱氢酶基因(ALDH1)转导的造血细胞对环磷酰胺的耐受性.以逆转录病毒载体pLXSN-ALDH1将醛脱氢酶基因导入人造血细胞系K562,应用PCR证实原病毒的整合,用RT-PCR检测ALDH1基因表达和用MTT法分析ALDH1过表达导致的4-氢过氧环磷酰胺的耐药表型.结果发现,逆转录病毒成功地介导了醛脱氢酶基因转移,全长ALDH1 cDNA以原病毒形式整合入受体K562细胞基因组,RT-PCR检测到ALDH1基因转录表达.ALDH1过表达的基因转移细胞对环磷酰胺的耐受性明显提高(4倍),IC50≈10 μmol/L.结论提示,体外ALDHl过表达足以引起对环磷酰胺耐受,为体内ALDH1基因转移以保护骨髓细胞免受环磷酰胺的毒性作用提供了实验依据.%The identification of genes inducing resistance to anticancer chemotherapeutic agents and their introduction into hematopoietic cells represents a promising approach to overcome bone marrow toxicity, the limiting factor for most high-dose chemotheapy regimens. Because resistance to cyclophosphamide has been correlated with increased levels of expression of the aldehyde-dehydrogenase(ALDH1 ) gene in tumor cells lines in vitro, this study tested whether ALDH1 overexpression could directly induce cyclophosphamide resistance. Results showed that a retroviral vector was used to transduce full-length human ALDH1 cDNA into human hematopoietic cell line K562 that was thel tested for resistance to 4-hydroxycyclophosphamide(4-HC), an active analogue of cyclophosphamide. Overexpression of the ALDH1 gene resulted in a significant increases in cyclophosphamide resistance in transduced K562 cells (50% inhibition concentration, IC50 ≈ 10 μmol/L). These findings indicate that ALDH1 overexpression is sufficient to induce cyclophosphamide resistance in vitro and provide a basis for testing the efficacy of ALDH1 gene transduction to protect bone marrow cells from high-dose cyclophosphamide in vivo.