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目的构建MDR1基因短发卡样RNA(shRNA)真核表达载体,观察对K562/A02人白血病细胞株MDR1基因的沉默作用以及对P-糖蛋白(P-gp)表达及功能的影响。方法以基因重组技术构建表达质粒,转染重组质粒pEGFP-C1/U6/MDR1-A和pEGFP-C1/U6/MDR1-B至K562/A02细胞株,通过半定量RT-PCR和蛋白质印迹法,检测MDR1基因表达及P-gp表达水平的变化；以MTT法检测阿霉素(ADM)对K562/A02细胞的半数抑制浓度(IC_(50))；高效液相色谱(HPLC)法检测细胞内ADM含量。结果构建的2种重组质粒pEGFP-C1/U6/MDR1-A和pEGFP-C1/U6/MDR1-B均明显抑制K562/A02细胞株MDR1基因表达,抑制率最高为48．2%±2．5%；同时抑制P-gp蛋白的表达,抑制率最高为50．67%。对ADM药物敏感性的相对逆转效率分别为40．8%和62．4%；同时使K562/ A02细胞内ADM含量增加。结论shRNA表达载体可明显抑制K562/A02细胞MDR1 mRNA的转录和P-gp蛋白的表达,增加K562/A02细胞内ADM含量,恢复K562/A02细胞对化疗药物的敏感性,逆转MDR1基因编码蛋白P-gp介导的多药耐药。
Objective To construct a short hairpin RNA (shRNA) eukaryotic expression vector of MDR1 gene and study its effect on the silencing of MDR1 gene and the expression and function of P-glycoprotein (P-gp) in K562 / A02 human leukemia cell line. Methods The recombinant plasmids pEGFP-C1 / U6 / MDR1-A and pEGFP-C1 / U6 / MDR1-B were transfected into K562 / A02 cell line by gene recombination technique. Semi-quantitative RT- The expression of MDR1 gene and the expression of P-gp were detected by MTT assay. The half inhibitory concentration (IC 50) of Adriamycin (ADM) on K562 / A02 cells was detected by MTT assay. ADM content. Results The two recombinant plasmids pEGFP-C1 / U6 / MDR1-A and pEGFP-C1 / U6 / MDR1-B all inhibited the expression of MDR1 gene in K562 / A02 cell line with the highest inhibitory rate of 48.2% ± 2.5 %; At the same time inhibit the expression of P-gp protein, the highest inhibition rate was 50.67%. The relative reversal efficiency of drug sensitivity to ADM was 40.8% and 62.4%, respectively. At the same time, ADM content in K562 / A02 cells increased. Conclusion The shRNA expression vector can significantly inhibit the transcription of MDR1 mRNA and the expression of P-gp protein in K562 / A02 cells, increase the content of ADM in K562 / A02 cells and restore the sensitivity of K562 / A02 cells to chemotherapeutic drugs, reverse the expression of MDR1 gene encoding protein P -gp-mediated multidrug resistance.