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

发布时间：2018-06-01 10:50

本文选题：血管生成素基因 + T细胞　；参考：《山东大学学报(医学版)》2016年12期

【摘要】：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。
[Abstract]:Objective to construct the lentivirus expression vector of human angiopoietin-1 (Ang-1) gene and to detect its expression in human umbilical cord mesenchymal stem cells (hUC-MSCs) and its effect on the immunosuppressive ability of human angiopoietin-1 (Ang-1) gene in human umbilical cord mesenchymal stem cells (hUC-MSCs). Methods Trizol was used to extract total RNAs from hUC-MSCs and reverse transcription-PCR was used to amplify the coding Ang-1 sequence into GV287 vector. The recombinant GV287-Ang-1 vector plasmid and lentivirus packaging plasmid pHelper 1.0 and p Helper 2.0 were co-transfected into 293T cells to collect the virus supernatant and purify and concentrate to determine the titer of the virus. The transfection efficiency was observed by fluorescence microscope and the expression of Ang-1 protein was detected by Western blotting. The proliferative activity of T lymphocytes was detected by CCK8 kit. Results the PCR products amplified by Ang-1 gene were consistent with the expected size. The recombinant lentivirus GV287-Ang-1 plasmid was sequenced by PCR and DNA. The results were consistent with the target gene sequence and the insertion direction was correct. The concentration and suspension titer of lentivirus was 2 脳 108TU/m L, and the best number of infection was that the Ang-1 expression in the 8.GV287-Ang-1 transfected group was significantly higher than that in the untransfected group and GV287 transfection group. The inhibition of T lymphocyte proliferation by hUC-MSCs with overexpression of Ang-1 was significantly higher than that of hUC-MSCs. Conclusion the lentivirus vector GV287-Ang-1 carrying Ang-1 gene can be successfully constructed, and can effectively transfect hUC-MSCs overexpression of Ang-1 protein, and can significantly improve the immunosuppressive ability of hUC-MSCs.
【作者单位】：山东大学齐鲁儿童医院儿科医学研究所;山东大学齐鲁医院低温医学研究室;山东大学齐鲁儿童医院血液肿瘤科;
【基金】：山东省自然科学基金(ZR2013HM001)
【分类号】：R457.7

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