论文部分内容阅读
目的:构建骨桥蛋白(OPN)特异性siRNA(smallin terfereRNA)真核表达载体,体外观察对OPN基因的沉默作 用.方法:采用基因克隆技术,将合成的特异性OPNRNA干 扰寡核苷酸序列插入真核表达载体mU6pro,构建OPNsiRNA 真核表达载体.以脂质体法将mU6pro空载体和2个 (mU6pro/OPN siRNA1和mU6pro/OPN siRNA2)重组质粒分 别导入具有高转移特性的GC9811胃癌细胞系.72h后用 RT PCR和Westernblotting技术检测各实验组胃癌细胞内 OPNmRNA及蛋白水平的表达情况.结果:成功构建OPN siRNA真核表达载体.转染OPN siRNA的胃癌细胞,72h后 OPNmRNA及蛋白表达下调,而以mU6pro/OPN siRNA1的抑 制效果更为明显.结论:构建的RNA干扰真核表达载体能明 显干扰OPNmRNA及蛋白的表达,为将其进一步应用于胃癌 的治疗研究奠定了基础.
OBJECTIVE: To construct an OPN-specific small interfering RNA (Eukaryotic expression vector) and observe the silencing of OPN gene in vitro.Methods: The specific OPN RNA interference oligonucleotide was inserted into the OPN gene by gene cloning The eukaryotic expression vector mU6pro was constructed and the mU6pro empty vector and two recombinant plasmids (mU6pro / OPN siRNA1 and mU6pro / OPN siRNA2) were respectively introduced into GC9811 gastric cancer cell line with high metastatic potential by liposome method. The expression of OPN mRNA and protein in gastric cancer cells were detected by RT-PCR and Westernblotting after 72 hours.Results: The OPN siRNA eukaryotic expression vector was successfully constructed.On OPN siRNA transfected gastric cancer cells, OPN mRNA and protein expression was down-regulated 72 h later, However, the inhibitory effect of mU6pro / OPN siRNA1 was more obvious.Conclusion: The RNA interference constructed Eukaryotic expression vector can significantly interfere with the expression of OPN mRNA and protein, which laid the foundation for its further application in the treatment of gastric cancer.