多药耐药基因反义寡核苷酸逆转肿瘤细胞耐药的初步研究

Reversal of drug resistance in multidrug resistant tumor cells by an oligomer complementary to the MDR1 gene

目的：克服肿瘤细胞的多药耐药（ＭＤＲ）。方法：用人工合成互补于ｍｄｒ１基因５′端转录起始部位的反义寡核苷酸（ＯＤＮ），直接转染耐药细胞株ＫＢ－８－５细胞，或以脂质体ｌｉｐｏｆｅｃｔｉｎ为载体进行基因转染实验，通过ＭＴＴ法检测细胞对柔红霉素（ＤＮＲ）的敏感性，流式细胞仪分析细胞内ＤＮＲ含量及免疫组化方法确定细胞表面糖蛋白（Ｐｇｐ）的表达水平。结果：ＯＤＮ可增加ＫＢ－８－５细胞内的ＤＮＲ浓度从而提高耐药细胞对ＤＮＲ的敏感性，ｌｉｐｏｆｅｃｔｉｎ进一步加强上述作用。在ＤＮＲ浓度为３．０ｍｇ／Ｌ组中，约有７４．４３％的被转染细胞对ＤＮＲ敏感而致死，基本达到药物敏感细胞株ＫＢ－３－１的水平。ＯＤＮ转染的ＫＢ－８－５细胞的Ｐｇｐ为弱阳性表达，低于阳性对照ＫＢ－８－５细胞的Ｐｇｐ表达水平。结论：ＯＤＮ的逆转作用可能与其互补结合的ｍｄｒ１ｍＲＮＡ降解或直接阻滞了Ｐｇｐ合成使其表达降低有关。

Objective:To overcome the multidrug resistance (MDR) in tumor cells. Methods:Human drug resistant cell line KB 8 5 was transfected with a synthetic oligodeoxynucleotide complementary to the 5′ end region of MDR1 gene (ODN).In vitro drug sensitivity was measured by MTT assay.Intracellular drug concentration was assessed by flow cytometric analysis and expression of P glycoprotein(Pgp) was determined by immunohistochemistry method.Results:The administration of ODN for 72 hours increased daunorubicin (DNR) accumulation and decreased Pgp expression in KB 8 5 cells.Incomplete reversal of MDR in the KB 8 5 cells was observed when lipofectin was used to deliver ODN to the cells. Lipofectin enhanced the reversing activity ,and approximately 74.43% of the cells lost their resistance to DNR.Conclusion: ODN circumvents resistance in the KB 8 5 cells may be ascribed to the reduced expression of Pgp in the cells.