慢病毒载体介导IL-24基因转导人骨髓间充质干细胞的实验研究

Study on Transduction of IL-24 Gene in Human Bone Marrow Mesenchymal Stem Cells by Lentiviral Vector

背景与目的以人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,hBMSCs)作为抑癌基因IL-24的细胞载体的研究目前未见报道。应用Gateway法构建共表达增强型绿色荧光蛋白(enhanced green uorescentprotein,EGFP)基因和IL-24基因的慢病毒载体,探讨其对hBMSCs的转导情况,为今后肿瘤的基因治疗奠定基础。方法应用DNA重组技术构建含有IL-24和EGFP基因的慢病毒表达载体,并与慢病毒包装系统(ViraPowerTMLentiviralPackaging Mix)共转染293FT细胞,收集上清,纯化浓缩,测定重组病毒的滴度。取重组慢病毒感染hBMSCs,通过嘌呤霉素筛选并纯化hBMSCs,应用实时荧光定量PCR(quantitative PCR,qPCR)及ELISA法分别检测hBMSCs中IL-24mRNA及IL-24蛋白水平的表达情况。结果成功构建了共表达IL-24和EGFP基因的重组慢病毒载体,经包装、纯化及浓缩,病毒滴度为7.25×107PFU/mL。重组慢病毒转导hBMSCs后,通过筛选获得纯化,转导效率可达到100%。qPCR检测示:转导组IL-24mRNA表达明显高于未转导组(P<0.05);ELISA法检测显示转导组hBMSCs上清液IL-24蛋白表达40g/L,未转导组为阴性。结论构建的携带IL-24基因的重组慢病毒载体可有效转导hBMSCs,表达IL-24蛋白。

[ Abstract ] Background and objective Up to know, no any study on using human bone marrow mesenchymal stem cells （hBMSCs） as cells carrier of tumor suppressor gene （IL-24） was reported. The aim of this study is to study the efficiency of transduction of hBMSCs by constructing the lentiviral vector in co-expressing enhanced green fluorescent protein （EGFP） gene and human IL-24 gene, and to lay a foundation for gene therapy of tumor in the future. Methods The lentivector which contain IL-24 and EGFP constructed by recombinant DNA technology were co-transfected to 293FT cells with ViraPowerTM Lentiviral Packaging Mix. The recombinant lentivirus infected with hBMSCs were selected and purified by puromycin. Expres- sion ofIL-24 mRNA and IL-24 protein levels were detected by real-time quantitative PCR （qPCR） and ELISA. Results ＂fhe recombinant lentiviral vector of co-expressing IL-24 gene and EGFP gene were successfully constructed by multisite Gateway technology3 virus can be packaged, purified and concentrated successfully, and the virus titer was 7.25x 107 PFU/mL. The ef- ficiency of recombinant lentivirus to transduce hBMSCs can reach 100% after selection. The result of qPCR showed that the level of IL-24 mRNA expression in transduced group was significantly higher than that in non-transduced group （P〈0.05）; ELISA detection confirmed that IL-24 protein expression of transduced group was positive in supernatant and the concentra- tion of IL-24 protein is 40 lag^L, while the non-transduced group was negative. Conclusion Lentiviral vector carrying recom- binant IL-24 gene can effectively transduce hBMSCs and express IL-24 protein.