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

发布时间：2018-04-26 03:10

本文选题：HT细胞 + MDR　；参考：《基因组学与应用生物学》2017年04期

【摘要】：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。
[Abstract]:HT9 cells of human acute promyelocytic leukemia (AHL) were used to interfere with the expression of MDR1 gene by using RNA interference RNAi technique to enhance the sensitivity of HT9 cells to paclitaxel. Three kinds of recombinant interference vectors targeting MDR1 gene were constructed. After stable transfection of HT9 cells, the expression levels of m RNA and protein of MDR1 gene were detected by qRT-PCR and Western blotting methods, and the sensitivity of transfected cells to paclitaxel was detected by MTT assay. The efflux function of the cells was detected by flow cytometry. The results showed that three recombinant interference vectors p3.1-1p3.1-2 and p3.1-3.3 were successfully constructed to inhibit the expression of MDR1 gene in HT9 cells. Among them, p3.1-3 had the best silencing effect on MDR1 gene. The silencing efficiency of m RNA and protein were 73.80% and 47.61%, respectively. Compared with the control group, the IC50 of the cells transfected with p3.1-3 recombinant interference vector decreased from 1.26 卤0.17 渭 g / mL to 0.46 卤0.07 渭 g / mL, the reversal rate was 63.48 卤5.91g / mL, and the efflux function of the drug was also significantly decreased. The results showed that all three recombinant interference vectors targeting MDR1 could inhibit the expression of MDR1 gene, among which p3.1-3 had the best effect on MDR1 gene interference, and could effectively enhance the sensitivity of HT9 cells to paclitaxel and reduce the drug efflux function of HT9 cells.
【作者单位】：齐齐哈尔大学生命科学与农林学院;
【基金】：黑龙江省自然科学基金(项目编号:C200624,C201241) 黑龙江省教育厅科学技术项目(项目编号:11511447,12511611);黑龙江省教育厅基本业务专项理工重点项目(项目编号:135109104) 齐齐哈尔大学2015年研究生创新科研项目(项目编号:YJSCX2015-024X)共同资助
【分类号】：R733.71

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