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目的 :探讨以多药耐药相关蛋白 (MRP)作为靶分子进行胃癌多药耐药基因治疗的可行性。方法 :采用已构建成功的mrp反义RNA真核载体pcDNA Amrp转染胃癌细胞系SGC790 1,用G418抗性筛选出稳定细胞克隆 ,命名为M SGC790 1;用32 P标记的寡核苷酸探针打点杂交检测M SGC790 1细胞中mrpmRNA表达的变化 ;从细胞生长曲线中 ,观察M SGC790 1细胞生长速度的变化 ;经 0 .0 0 5 μg/ml的长春新碱 (VCR)处理 1月后 ,行流式细胞术检测M SGC790 1细胞周期的改变 ;同时 ,行MTT法检测M SGC790 1对VCR的IC50 值 ;最后 ,行Habit法检测M SGC790 1中谷胱苷肽S 转移酶 (GST)活性。结果 :①M SGC790 1细胞的mrpmRNA表达的阳性信号明显较对照组弱。②M SGC790 1细胞生长速度与对照组无明显差异 (P >0 .0 5 )。③M SGC790 1生长明显阻滞于S期 (P <0 .0 5 )。④M SGC790 1对VCR的IC50 值显著降低 (P <0 .0 5 )。⑤M SGC790 1中GST活性与对照组相比无明显差异 (P >0 .0 5 )。结论 :本文为以mrp为靶基因进一步进行胃癌多药耐药基因治疗的研究奠定了一定的实验基础
Objective: To explore the feasibility of multidrug resistance gene therapy for gastric cancer using multidrug resistance-associated protein (MRP) as a target molecule. METHODS: The human gastric cancer cell line SGC790 was transfected with the constructed mrp antisense RNA eukaryotic vector pcDNA Amrp 1 and a stable cell clone was selected by G418 resistance, named as M SGC790 1; the 32 P-labeled oligonucleotide was used for detection. The changes of mrpm RNA expression in M SGC790 1 cells were detected by dot blot hybridization; the growth rate of M SGC790 1 cells was observed from the cell growth curve; after treated for one month with 0.050 μg/ml vincristine (VCR). Flow cytometry was used to detect the change of cell cycle of M SGC790 1. At the same time, the IC50 value of M SGC790 1 against VCR was detected by MTT assay. Finally, the activity of glutathione S transferase (GST) in M SGC790 1 was detected by Habit assay. . Results : The positive signal of mrpm RNA expression of 1M SGC790 1 cells was significantly weaker than that of the control group. The growth rate of 2M SGC790 1 cells was not significantly different from that of the control group (P > 0.05). The growth of 3M SGC790 1 was significantly arrested in S phase (P < 0.05). The IC50 value of 4M SGC790 1 on VCR was significantly lower (P < 0.05). GST activity in 5M SGC790 1 was not significantly different from the control group (P > 0.05). Conclusion : This study lays a foundation for the further study of multidrug resistance gene therapy for gastric cancer using mrp as a target gene.