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本研究以A549/DDP细胞为实验对象,利用shRNA(short hairpin RNA)沉默MDR1基因,逆转人肺癌A549/DDP细胞对顺铂的耐药性。构建3种靶向MDR1基因重组干扰载体,稳定转染A549/DDP细胞,qRT-PCR检测MDR1 mRNA表达水平,Western blotting检测MDR1蛋白表达水平,MTT法检测细胞对顺铂的敏感性。结果显示成功构建了3种靶向MDR1的重组表达载体p2.1-1、p2.1-2和p2.1-3。3种干扰表达载体均能有效沉默A549/DDP细胞MDR1基因表达,其中p2.1-3对MDR1沉默效果最好,对mRNA和蛋白的沉默效率分别为51.47%和53.24%。转染p2.1-3的细胞对顺铂的IC50由(72.08±7.00)μmol/L降至(31.89±3.39)μmol/L,逆转率达到(67.60±5.70)%。这些结果表明靶向MDR1的重组干扰载体均能够有效抑制MDR1表达,其中p2.1-3干扰效果最佳并且能逆转A549/DDP细胞对顺铂的耐药性。
In this study, A549 / DDP cells as experimental subjects, the use of shRNA (short hairpin RNA) silencing MDR1 gene, reversal of human lung cancer A549 / DDP cells resistance to cisplatin. Three MDR1 gene-targeting recombinant plasmids were constructed and stably transfected into A549 / DDP cells. The expression of MDR1 mRNA was detected by qRT-PCR. The protein expression of MDR1 was detected by Western blotting. The sensitivity of cells to cisplatin was determined by MTT assay. The results showed that MDR1 gene expression in A549 / DDP cells was successfully silenced by three kinds of expression vectors of p2.1-1, p2.1-2 and p2.1-3 p2.1-3 had the best silencing effect on MDR1, and its silencing efficiency on mRNA and protein was 51.47% and 53.24% respectively. The IC50 of cells transfected with p2.1-3 decreased from (72.08 ± 7.00) μmol / L to (31.89 ± 3.39) μmol / L, and the reversal rate reached 67.60 ± 5.70%. These results indicate that the recombinant interfering vector targeting MDR1 can effectively inhibit the expression of MDR1, wherein p2.1-3 has the best interference effect and can reverse the drug resistance of A549 / DDP cells to cisplatin.