人血管生成素1基因慢病毒表达载体的构建及其在脐带间充质干细胞的表达

Construction and identification of lentiviral vector for angiopoietin-1 gene and its expression in human umbilical cord mesenchymal stem cells

目的通过基因重组技术构建人血管生成素-1(Ang-1)基因慢病毒表达载体,并检测其在人脐带间充质干细胞(hUC-MSCs)的表达及对hUC-MSCs免疫抑制能力的影响。方法应用Trizol法从hUC-MSCs提取总RNA,反转录获取c DNA,PCR扩增获得编码Ang-1的序列克隆到GV287载体中。将重组GV287-Ang-1载体质粒和慢病毒包装质粒pHelper 1.0和p Helper 2.0共转染293T细胞,收集病毒上清,纯化浓缩测定病毒滴度。采用荧光显微镜观察转染效率,Western blotting法检测Ang-1蛋白表达,并通过CCK8试剂盒检测T淋巴细胞增殖活性。结果 Ang-1基因扩增PCR产物与预期大小一致。重组慢病毒GV287-Ang-1质粒经PCR和DNA测序分析显示,所得结果与目的基因序列一致且插入方向正确。包装慢病毒浓缩悬液滴度为2×108TU/m L,最佳感染复数为8。GV287-Ang-1转染组细胞Ang-1表达显著高于未转染组和GV287转染组。过表达Ang-1的hUC-MSCs对T淋巴细胞的增殖抑制显著高于单纯的hUC-MSCs。结论成功构建携带Ang-1基因的慢病毒载体GV287-Ang-1,并可有效转染hUC-MSCs过表达Ang-1蛋白,且能显著提高hUC-MSCs的免疫抑制能力。

Objective To construct the lentivirus vector carrying angiopoietin-1 gene（ Ang-1）,and to investigate its expression in human umbilical cord mesenchymal stem cells（ hUC-MSCs） and the immunomodulatory effect on Ang-1-modified hUC-MSCs. Methods The c DNA encoding full-length human Ang-1 was amplified by RT-PCR from the total RNA of hUC-MSCs,and then subcloned to lentiviral vector backbone plasmid GV287. The recombinant plasmid GV287-Ang-1 was co-transfected with packaging and enveloping plasmids p Helper 1. 0 and p Helper 2. 0 into 293 T cells. Vector titer was quantified using cell fluorescence. The transfection was detected by fluorescence microscopy,and protein expression of Ang-1 was detected by Western blotting. T lymphocytes proliferation was assessed by cell counting kit 8（ CCK8）. Results The recombinant lentiviral vector GV287-Ang-1 was verified correctly by PCR and DNA sequencing. The titer of concentrated virus was 2 × 10^8 TU / mL,and the optimal MOI for transfecting hUC-MSCs was 8.Western blotting results showed that the Ang-1 expression of the GV287-Ang-1 transfected group was significantly higherthan those of the non-transfected group and GV287 group. The suppression ratio of T lymphocytes in Ang-1-UC-MSCs group was significantly increased as compared to that in the UC-MSCs group. Conclusion The lentivirus vector carrying human Ang-1 was constructed successfully and overexpressed effectively in hUC-MSCs. Ang-1-modified hUCMSCs can enhance the immunosuppressive capability,which suggests a potential clinical application.