Cyclin E分子siRNA慢病毒载体的构建及其对骨肉瘤细胞系Sosp9607的影响

Construction of SiRNA Lentivirus Vector and the Influence on Sosp9607

设计筛选针对人cyclin E分子的siRNA序列,构建相应的siRNA慢病毒载体,检测其对骨肉瘤细胞系Sosp9607胞内cyclin E分子表达水平的影响。首先设计并合成4对siRNA双链寡聚核苷酸,与cyclin E分子真核表达载体共同转染293T细胞。挑选出最有效抑制cyclin E表达的序列,应用基因工程技术,将该序列连接于慢病毒载体pLKO.1中,构建携带针对目的基因cyclin E的siRNA慢病毒载体pLKO-CE。使用慢病毒包装质粒混合物和构建好的慢病毒载体共转染293T细胞,收获病毒上清,感染Sosp9607细胞系,对病毒感染后抑制cyclin E表达的效果进行检测。结果在4条针对cyclin E分子设计的siRNA序列中,siRNA-464最为有效的抑制了外源性cyclin E分子的表达;带有该序列的慢病毒载体pLKO-CE可以完全抑制Sosp9607细胞中天然分子的表达,引起Sosp9607细胞生物学行为的改变:G1期细胞增多,S期减少,细胞增殖受抑制。说明应用基因工程技术成功构建了针对cyclin E分子的RNA干扰慢病毒载体,可有效抑制骨肉瘤细胞cyclin E的表达,使肿瘤细胞增殖减缓,为深入研究针对cyclin E的基因治疗方案的临床前实验研究提供了实验证据和理论依据。

To construct a lentiviral vector of RNA interference （RNAi） of human cyclin E gene, the overexpression vector pcDNA—cyclin E was confirmed in our previous study. 4 sequence of siRNA targeting human cyclin E gene was designed,synthesized and co-transfected into 293T cells to screen the effective one. The complementary DNA containing both sense and antisense Oligo DNA of the effective sequence siRNA—464 was cloned into the pLKO.1 vector. The resulting lentiviral vector containing cyclin E shRNA was named pLKO—CE and it was confirmed by restriction endonuclease analysis and sequencing. 293T cells were co-transfected with lentiviral vector pLKO—CE,VSV—G and Δ8.9. All virus stocks were produced by calcium phosphate mediated transfection,and the supernatant of transfected 293T cells was collected to obtain the recombinant lentivirus. The interference effective of virus was tested according to the expression level of cyclin E on Sosp9607 cells.It is resulted： that PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of cyclin E producing shRNA was constructed successfully,and which can inhibit the expression of cyclin E molecule in Sosp9607 cell effectively. It is concluded that lentivirus RNAi vector of cyclin E is constructed successfully.