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本研究旨在构建并筛选MDR1序列特异性短发夹状RNA干扰载体,研究其对K562/A02细胞的影响。采用脂质体介导方法,将含mdr1-sh RNA质粒转入K562/A02细胞,经G418筛选得到稳定表达细胞株,用实时定量RT-PCR和Western blot分别检测干扰后mdr1 mRNA水平及蛋白水平的表达变化,通过CCK8测定干扰后细胞对化疗药的敏感性变化,Rhodamine123外排实验检测P-gp功能的变化。结果发现:与对照组相比,4种MDR1-sh RNA载体均能显著下调P-gp的表达,其中508-526靶位点的shRNA作用效果最好。结论:筛选得到的MDR1-sh RNA载体能够显著下调MDR1的表达,逆转耐药表型,为后续进行的动物实验研究创立了条件。
The purpose of this study was to construct and screen MDR1 sequence-specific short hairpin RNA interference vectors and study its effect on K562 / A02 cells. The plasmid containing mdr1-sh RNA was transfected into K562 / A02 cells by lipofectamine. The stable cell lines were screened by G418. The mdr1 mRNA and protein levels were detected by real-time quantitative RT-PCR and Western blot respectively The changes of cell chemosensitivity to chemotherapeutic drugs were determined by CCK8 assay. The changes of P-gp function were detected by Rhodamine123 efflux assay. The results showed that: compared with the control group, all four MDR1-sh RNA vectors could significantly down-regulate the expression of P-gp, and the shRNA effect of the target site 508-526 was the best. CONCLUSION: The screened MDR1-sh RNA vector can significantly down-regulate the expression of MDR1 and reverse the drug resistance phenotype, creating the conditions for subsequent animal experiments.