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目的:构建人耐药白血病细胞多药耐药基因-1小干扰RNA并研究其功能。方法:人工合成编码mdr1小发夹状双链RNA的DNA片段,与pSilencer4.1-CMV质粒连接构建RNAi真核表达载体,采用脂质体介导法转染人耐药白血病细胞K562/A,经潮霉素B筛选转基因阳性克隆细胞,RT-PCR和Western Blotting检测转基因细胞中mdr1基因的表达量,MTT法检测转基因细胞对阿霉素的敏感性。结果:RT-PCR结果显示,与未转基因组和阴性对照组比较,RNA干扰组mdr1基因在mRNA水平上表达量降低43.55%;Western Blotting检测显示,RNA干扰组mdr1基因在蛋白水平上表达量降低69.46%;MTT法检测显示mdr1干扰细胞对化疗药物柔红霉素的敏感性提高23倍。结论:mdr1小发夹状RNA可显著抑制K562/A细胞中的mdr1基因的表达,提高白血病细胞对化疗药物的敏感性,对白血病多药耐药性的逆转和白血病的治疗具有重要理论和实际意义。
Objective: To construct a multi-drug resistant gene-1 small interfering RNA of human multidrug resistant leukemia cells and study its function. Methods: A DNA fragment encoding mdr1 small hairpin double stranded RNA was synthesized and ligated with pSilencer4.1-CMV plasmid to construct RNAi eukaryotic expression vector. The recombinant plasmid was transfected into K562 / A human leukemia cell line by lipofectamine. Transgenic positive clones were screened by hygromycin B. The expression of mdr1 gene in transgenic cells was detected by RT-PCR and Western Blotting. The sensitivity of transgenic cells to doxorubicin was detected by MTT assay. Results: Compared with untransformed group and negative control group, the mRNA expression of mdr1 in the RNAi group decreased by 43.55% compared with the untransformed group and the negative control group. Western Blotting showed that the mdr1 gene expression in the RNAi group decreased at the protein level 69.46%; MTT assay showed mdr1 interfering cells increased chemosensitivity to daunorubicin 23-fold. Conclusions: Small hairpin RNA of mdr1 can significantly inhibit the expression of mdr1 gene in K562 / A cells, increase the sensitivity of leukemic cells to chemotherapeutic drugs, reversal of multidrug resistance of leukemia and treatment of leukemia have important theoretical and practical significance.