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目的探讨RNAi技术经慢病毒载体介导的siRNA对Jiyoye细胞c-myc基因表达的抑制作用。方法设计并合成RNA干扰序列,退火后连接到pLVX干扰载体上,构建PLVX-c-myc表达载体,经慢病毒介导转染人Jiyoye细胞株培养72 h。实验分为空白对照(未转染)、c-myc-neg、c-myc-1、c-myc-2、c-myc-3组。采用流式细胞术检测各组细胞转染率,采用real-time PCR和Western blot法检测各组细胞c-myc mRNA及蛋白表达水平的变化。结果构建pLVX-c-myc-neg、pLVX-c-myc-1、pLVX-c-myc-2、pLVX-c-myc-3重组表达载体。与c-myc-neg组相比,c-myc-1、c-myc-2和c-myc-3组c-myc mRNA及蛋白表达水平均明显下调(P<0.05),以c-myc-3组下降最显著(P<0.05)。结论成功构建c-myc shRNA表达载体,利用慢病毒介导转染Jiyoye细胞的c-myc基因表达下调,为进一步探讨沉默c-myc基因在白血病和淋巴瘤靶向治疗中的作用奠定了实验基础。
Objective To investigate the inhibitory effect of RNAi on c-myc gene expression in Jiyoye cells mediated by lentiviral vectors. Methods The RNA interference sequence was designed and synthesized. After annealing, the recombinant plasmid was ligated into pLVX vector and constructed PLVX-c-myc vector. The recombinant plasmid was transfected into human Jiyoye cell line for 72 h by lentivirus. The experiment was divided into blank control (untransfected), c-myc-neg, c-myc-1, c-myc-2 and c-myc-3 groups. The transfection efficiency of each group was detected by flow cytometry. The expression of c-myc mRNA and protein in each group was detected by real-time PCR and Western blot. Results The recombinant plasmids pLVX-c-myc-neg, pLVX-c-myc-1, pLVX-c-myc-2 and pLVX-c-myc-3 were constructed. Compared with c-myc-neg group, c-myc-1, c-myc-2 and c-myc-3 c-myc mRNA and protein expression levels were significantly decreased (P < The three groups decreased the most significantly (P <0.05). Conclusion The c-myc shRNA expression vector was constructed successfully and the down-regulation of c-myc gene expression by lentivirus-mediated Jiyoye cells laid the foundation for further study on the role of silencing c-myc gene in leukemia and lymphoma targeting therapy .