干扰MDR1基因表达提高急性早幼粒白血病HT9细胞对紫杉醇的敏感性

Interfering the Expression of MDR1 Gene Might Enhance the Sensitivity of Acute Promyelocytic Leukemia HT9 Cells to Paclitaxel

以人类急性早幼粒白血病HT9细胞为实验对象,利用RNA干扰(RNA interference,RNAi)技术干扰MDR1基因表达,提高HT9细胞对紫杉醇的敏感性。构建了3种靶向MDR1基因的重组干扰载体,稳定转染HT9细胞后,采用qRT-PCR和Western blotting方法,分别检测MDR1基因的m RNA和蛋白表达水平,用MTT法检测各转染组细胞对紫杉醇的敏感性,流式细胞术检测各组细胞药物外排功能。结果显示,成功构建了3种靶向MDR1的重组干扰载体p3.1-1、p3.1-2和p3.1-3。3种重组干扰载体不同程度抑制HT9细胞中MDR1基因的表达,其中重组干扰载体p3.1-3对MDR1基因沉默效果最好,对m RNA和蛋白的沉默效率分别为73.80%和47.61%,与对照组相比,转染p3.1-3重组干扰载体的细胞对紫杉醇的IC50由(1.26±0.17)μg/m L降至(0.46±0.07)μg/m L,逆转率达到(63.48±5.91)%,并且药物外排功能也明显降低。以上实验结果表明,3种靶向MDR1的重组干扰载体均能抑制MDR1基因表达,其中p3.1-3对MDR1基因干扰效果最好,并且能够有效提高HT9细胞对紫杉醇的敏感性,降低细胞药物外排功能。

The acute promyelocytic leukemia HT9 cells were used as the main experimental subject. RNA interference technology was used to interfere the expression of MDR1 to enhance the sensitivity of HT9 cells to paclitaxel. Three recombinant enterfering vectors target to MDR1 were bulit and transfected into HT9 cells. The expression level of MDR1 was detected by qRT-PCR and Western blotting assay. MTT and flow cytometry were adopted to detect the sensitivity to paclitaxel and the drug effius capatity of transfected cells. The results showed that three recombinant vectors target to MDR1 named p2.1-1, p2.1-2 and p2.1-3 were built successfully. In addition, the three recombinant interfering vectors inhibited the expression ofMDR1 gene in HT9 cells to different degrees, among which p2.1-3 had the best gene silencing effect on MDR1 gene, and its silence efficiency to mRNA and protein was 73.80% and 47.61% respectively. Compared to the control group, the IC50 ofMDR1 gene to paclitaxel decreased from （1.26±0.17） μg/mL to （0.46±0.07） μg/mL after transfected by p2.1-3, the reversal rate reached （67.60±5.70）%, and the drug effiux capacity of the cells also reduced. These experimental results indicated that the three recombinant interference vectors targeting MDR1 gene all could inhibit the expression of MDR1 gene, in which p2.1-3 had the best interfering effect and could effectively enhance the sensitivity of HT9 cells to paclitaxel and reduce the drug effiux capacity of cells.