shRNA沉默多药耐药胃癌细胞7901/VCR MDR1基因表达的实验研究

目的利用RNAi技术干扰胃癌多药耐药细胞（7901/VCR）多药耐药基因（MDR1）的表达,为临床肿瘤的基因治疗奠定基础。方法根据MDR1的碱基序列设计并合成两对短发夹RNA,构建重组载体,用阳离子脂质体法体外转染7901/VCR;采用MTT法检测转染胃癌细胞的生长抑制率,流式细胞术检测细胞周期变化,RT-PCR和Western blot检测转染前后MDR1 mRNA和蛋白表达的变化。结果成功构建RNAi质粒载体,转染后细胞IC50明显降低（P〈0.05）;G1期细胞增加,S期细胞减少（P〈0.05）;MDR1 mRNA转录水平下降（P〈0.05）,P-糖蛋白（P-gp）的表达水平降低（P〈0.05）。结论RNAi能明显抑制胃癌细胞7901/VCR MDR1mRNA和P-gp蛋白的表达,进而对肿瘤细胞多药耐药性有明显的逆转作用,为基因治疗提供了一种新的手段。

Study on silencing expression of MDR1 gene in multidrug resistance gastric cancer cell 7901/VCR by shRNA

Objective To investigate the expression of MDR1 gene in multidrug resistance gastric cancer cell 7901/VCR by RNA interference,in order to lay the foundation for clinical tumorous gene therapy. Methods The recombinant vectors of shRNA aiming at two MDR1 gene sequences were constructed and used to transfect the cell line 7901/VCR by LipofectamineTM 2000. The inhibition rate of cell proliferation was examined by MTT method. The ceil cycle was detected by flow cytometry. The mRNA of MDR1 gene was identified by RT-PCR and the protein expression of MDR1 gene was detected by Western blot. Results The RNAi vectors were successfully constructed. After transfection,cell IC50 was greatly reduced （ P 〈 O. 05）;S phase cells were reduced and G1 phase cells increased （P〈 0. 05）; the expressions of MDR1 mRNA and P-gp were reduced （P〈0.05）. Conclusions RNAi can significantly inhibit MDR1 mRNA and P-gp expression in gastric cancer cell 7901/VCR, and reverse tumor multidrug resistance. Therefore it may provide a new gene therapeutic method.