血管生长素-1基因慢病毒载体的构建及其在间质干细胞的表达

Construction of lentiviral vector carrying the Ang-1 gene and its expression in the rMSCs

目的:构建带有血管生长素-1(Ang-1)基因的慢病毒载体并实现该基因在大鼠骨髓间质干细胞(rMSCs)中的表达。方法:RT-PCR获得大鼠Ang-1基因,限制性内切酶酶切和基因重组构建慢病毒载体质粒PNL-Ang-1-IRES2-EGFP,在脂质体介导下与包装质粒HELPER,包膜质粒VSVG共转染293T细胞包装生产慢病毒颗粒并感染rMSCs。PCR检测Ang-1基因的插入,细胞RT-PCR检测Ang-1mRNA表达,免疫细胞化学和Western blotting检测Ang-1蛋白的表达。结果:所获Ang-1基因经测序证实与GenBank报道的序列一致。慢病毒载体质粒PNL-Ang-1-IRES2-EGFP经SaⅠl和BamHⅠ双酶切后电泳显示1 512 bpAng-1目的基因片段和10.5 kb PNL-IRES2-EGFP载体片段。病毒基因组PCR证实Ang-1基因插入。荧光显微镜观察到EGFP表达。细胞RT-PCR检测到Ang-1mRNA表达。免疫细胞化学和Western blotting检测到Ang-1蛋白表达。结论:成功构建带有Ang-1基因的慢病毒载体并实现在rMSCs中的表达,为Ang-1基因修饰干细胞的应用研究奠定了基础。

AIM ： To construct lentiviral vector carrying the angiopoietin - 1 （Ang - 1 ） gene, and make it express Ang -1 in the rat mesenchymal stem cells （rMSCs）. METHODS： The cDNA encoding the CDS of Ang -1 gene was obtained from the placenta of the adult Fisher 344 rats with RT - PCR. After digestion with restrication endonuclease, the Ang - 1 gene was recombined to construct the transfer plasmid PNL - Ang1 - IRES2 - EGFP. The three - plasmid system of lentiviral vector was consisted of PNL - Ang1- IRES2 - EGFP, the packaging plasmid HELPER, and the envelope plasmid VSVG, which were co -transfected to 293T cells mediated by lipofectamin2000 to produce lentiviral particles. The rMSCs were infected by obtained lentiviral particles. The insertion of Ang - 1 gene was detected by PCR, the mRNA expression of Ang - 1 in rMSCs was detected with RT - PCR, the protein expression of Ang - 1 was observed with immunocytochemistry and Western blotting methods. RESULTS： The result of sequencing showed that the cloned Ang - 1 gene was consistent with the sequence reported in GenBank. After digestion with restrication endonuclease, the 1 512 bp fragment of Ang - 1 gene and the 10. 5 kb vector fragment of PNL - IRES2 - EGFP were observed with gel electrophoresis. The insertion of Ang -1 gene in viral genome was confirmed. The EGFP expression was observed with the fluorescent microscope. In infected rMSCs, the mRNA and protein expressions of Ang - 1 were confirmed. CONCLUSION ： Lentiviral vector carrying Ang - 1 gene has been successfully constructed. The infected rMSCs are able to express the Ang - 1 mRNA and Ang - 1 abundantly. This will facilitate the following exploratory development of Ang - 1 gene - modified rMSCs.