摘要
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, we used A549/DDP cells as experimental subjects, and used shRNA （short hairpin RNA, shRNA） silence MDR1 gene to reverse the drug resistance of human lung cancer A549/DDP cells to cisplatin. We constructed three targeting MDR1 genes recombinant interference vector, stably transfected A549/DDP cells, used qRT-PCR to detect the mRNA expression level of MDR1, used Western blotting to detect the protein expression level ofMDR1, and used MTT to detect the sensitivity of cells to cisplatin. The results showed that we successfully constructed three recombinant expression vectors p2.1-1, p2.1-2 and p2.1-3 of targeting MDR1 gene. These three interference expression vectors all could effectively silence the MDR 1 gene expression of A549/DDP cells. Among them, p2.1-3 had the best silencing effect on the expression of MDR1 and the silencing efficiency of mRNA and protein were 51.47% and 53.24% respectively. The IC50 value of cells of transfected p2.1-3 to cisplatin reduced from （72.08±7.00） μmol/L to （31.89±3.39） μmol/L and the reversal rate increased to （67.60±5.70）%. These results showed that the targeting MDR1 of recombinant interference vectors could effectively inhibit the expression of MDR1. The interference effect of p2.1-3 was the best and could reverse the drug resistance of A549/DDP cells to cisplatin.
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2016年第12期3219-3223,共5页
Genomics and Applied Biology
基金
黑龙江省自然科学基金(批准号:C200624
C201241)
黑龙江省教育厅科学技术项目(批准号:11511447
12511611)
齐齐哈尔大学2015研究生创新科研项目(YJSCX2015-024X)共同资助
