pSiRNA-Dll4逆转录病毒载体的构建及对骨肉瘤UMR106细胞Dll4蛋白表达的作用

Construction of pSiRNA-Dll4 recombinant retroviral vector and its effect on UMR106 cells in vitro

目的建立逆转录病毒介导的人Dll4基因RNA干扰体外表达体系并观察对骨肉瘤UMR106细胞的作用。方法将人Dll4基因RNAi双链转录DNA片段重组到逆转录病毒质粒psilencer5.1-H1Retro中,构建成携带人Dll4基因RNAi逆转录病毒载体pSiRNA-Dll4,经PT67细胞包装后,产生的重组逆转录病毒感染骨肉瘤细胞UMR106,用Westernblot分别检测对转染细胞人Dll4蛋白表达的影响。结果重组pSiR-NA-Dll4质粒经测序鉴定正确。重组逆转录病毒滴度可达221×104cfu/mL,感染UMR106骨肉瘤细胞3d能明显抑制细胞Dll4蛋白表达,其表达水平明显低于阴性对照组和未干扰组。结论成功构建了沉默Dll4表达的载体pSiRNA-Dll4,并且该载体能有效地敲低骨肉瘤细胞Dll4基因表达。

[ Objective] To construct a retrovirus-mediated expression system containing double strands DNA for RNA interference on human Dll4 gene and study its inhibitory effect on human cell lines UMR106 in vitro. [Methotis] A recombinant retroviral vector pSiRNA2Notehl was generated by cloning a double strands DNA for RNA in- terference on human Notcht-gene into a retroviral vector psilencer 5. 1-H1 Retro. Human glioma cell lines Osteosarcoma were infected with the viral supematant from the FF67 clones. After 3 d, the viability, Notchl mRNA and p rotein of the transfected cells were examined by Western blot. [Results] The pSiRNA-Dll4 recombinant retroviral vector had been constructed correctly. The titer assayed on NIH3T3 cells was up to 221×10^4 cfu/mL Three days after transfection, the viability, Dl14 protein of the transfected cells decreased significantly. [Conclusions] Dll4 siRNA exp ression vector pSilence Dll4 is successfully constructed that can significantly knock down Dll4 gene expression in osteosarcoma ceils., with potential utility in the gene therapy for human malignant Osteosarcoma.