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目的:采用化学修饰的小干扰RNA(siRNA)在体内外抑制含激酶插入区受体(KDR)基因表达,探讨化学修饰的siRNA介导的RNA干扰(RNA i)技术在乳腺癌基因治疗的可行性和特异性。方法:采用阳离子脂质体L ipofectam ine2000TM作为转染试剂将针对人KDR基因的siRNA转染人类乳腺细胞株MCF-7,诱导RNA i,采用四甲基偶氮唑蓝(MTT)法,RT-PCR,Western blot试验等检测KDR基因和蛋白表达及细胞增殖变化。采用阳离子聚合物纳米粒In vivojetPEITM为转染试剂将siRNA直接注射进裸鼠移植瘤,监测肿瘤生长变化,RT-PCR,免疫组化方法等监测KDR基因和蛋白表达变化。结果:靶向KDR基因siRNA转染MCF-7后,细胞增殖被抑制,KDRmRNA和蛋白的表达明显降低;裸鼠体内实验显示siRNA治疗组瘤组织的增长受到明显抑制;RT-PCR,免疫组化结果同时表明治疗组KDR表达下调。各对照组指标无明显变化。结论:化学修饰的siRNA介导的RNAi在体内外均能成功抑制靶基因的表达和MCF-7细胞增殖,是潜在的肿瘤治疗新方法,而KDR亦可作为肿瘤治疗的新靶点。
OBJECTIVE: To investigate the effect of chemically modified siRNA-mediated RNA interference (RNAi) on the gene therapy of breast cancer in vitro and in vivo, using chemically modified small interfering RNA (siRNA) to inhibit KDR gene expression in vitro and in vivo Sexual and specific. Methods: siRNA targeting human KDR gene was transfected into human breast cell line MCF-7 using cationic liposome L ipofectam ine 2000 TM as transfection reagent. RNAi was induced by MTT method and RT- PCR, Western blot test KDR gene and protein expression and cell proliferation changes. Injecting siRNA into nude mice xenografts with transfection reagent using cationic polymer nanoparticle In vivojet PEITM to monitor tumor growth, RT-PCR, immunohistochemical methods to monitor KDR gene and protein expression changes. Results: The proliferation of MCF-7 cells transfected with siRNA targeting KDR gene was inhibited and the expression of KDR mRNA and protein was significantly decreased. The in vivo experiments in nude mice showed that the growth of siRNA group was significantly inhibited. The results also showed that KDR expression was down-regulated in the treatment group. The control group indicators no significant change. CONCLUSION: Chemically modified siRNA mediated RNAi successfully inhibits target gene expression and MCF-7 cell proliferation both in vitro and in vivo and is a potential new therapeutic approach for tumor therapy. However, KDR can also serve as a new target for tumor therapy.