构建能转染人骨髓间充质干细胞的Ad5 GM-CSF-IL-2腺病毒载体

背景：如何将树突状细胞和T细胞活化因子运送至肿瘤局部是有待解决的难题之一。利用人骨髓问充质干细胞具有肿瘤趋向性和低免疫原性的特性，将树突状细胞和T细胞活化因子导入骨髓间充质干细胞后运送至肿瘤局部表达可能是解决上述难题的方案之一。目的：构建共表达粒细胞-巨噬细胞集落刺激因子和白细胞介素2的5型腺病毒载体（Ad5GM-CSF-IL-2），感染人骨髓间充质干细胞，检测粒细胞一巨噬细胞集落刺激因子和白细胞介素2的表达水平和持续时间，为体内活化树突状细胞和T细胞提供实验依据。方法：提取人外周血单个核细胞总RNA，以此为模板用RT-PCR方法扩增粒细胞-巨噬细胞集落刺激因子和白细胞介素2基因cDNA，PCR扩增片段分别插入pcDNA3．1／Myc-His（-）B真核表达载体中，再利用脑心肌炎病毒内部核酸进入位点（IRES）序列，构建腺病毒载体穿梭质粒pDC515GM-CSF-IRES-IL-2。采用AdMax^TMFLP重组腺病毒载体体系，将穿梭质粒pDC515GM-CSF-IRES-IL-2与pBGHfrt△E1，3FLP骨架质粒共转染至293细胞，通过重组酶位点特异性重组获得Ad5GM-CSF-IL-2。Ad5GM-CSF-IL-2感染人骨髓间充质干细胞后经v射线照射使其失去增殖能力，分别用粒细胞-巨噬细胞集落刺激因子和白细胞介素2ELISA试剂盒在不同的时间点检测细胞上清中粒细胞-巨噬细胞集落刺激因子和白细胞介素2蛋白的水平。结果与结论：经测序证实，克隆获得的粒细胞-巨噬细胞集落刺激因子、白细胞介素2的cDNA序列分别与GenBank NM_000758（435bp）和GenBank NM_000586（462bp）提供的序列完全一致。用AdMax^TMFLP重组腺病毒载体可高效、快捷地获得所要包装的腺病毒载体，通过IRES连接粒细胞-巨噬细胞集落刺激因子和白细胞介素2两个基因，均获得高效表达。Ad5GM-CSF-IL-2感染人骨髓间充质干细胞后可连续7d高水平地分泌粒细胞-巨噬细胞集落刺激因子和白细胞介素2。

Construction of an adenoviral vector encoding Ad5GM-CSF-IL-2 in human bone marrow mesenchymal stem cells

BACKGROUND： A problem needed to be solved is how to deliver activating factors for dendritic cells and T cells into the tumors. Owing to the characteristics of tumor tropism and weak immunogenicity, human bone marrow mensenchymal stem cells are used as a vehicle for transferring the activating factor genes of the dendritic cells and T cells to the tumor, which may be a protocol to solve the problem. OBJECTIVE： To construct a type 5 adenoviral （Ad） vector encoding granulocyte-macrophage colony stimulating factor （GM-CSF） and interleukin-2 （IL-2） genes, and detect the expression levels and duration of GM-CSF and IL-2 after infection of human bone marrow mesenchymal stem ceils, providing experimental evidence for in vivo activation of dendritic cells and T cells. METHODS： GM-CSF and IL-2 cDNAs from the total RNA extracted by human peripheral blood mononuclear cells were cloned by RT-PCR and inserted into the eukaryotic expression vector pcDNA3.11Myc-His（-）B. GM-CSF and IL-2 cDNAs linked by the internal ribosomal entry sites （IRES） from encephalomyocarditis virus were subcloned into the shuttle vector pDC515 for the construction of pDC515 GM-CSF-IRES-IL-2. By using AdMaxTM adenovirus vector system, the shuttle vector pDC515 GM-CSF-IRES-IL-2 and the backbone vector pBGHfrt△E1,3 FLP were cotransfected into 293 ceils, and Ad5 GM-CSF-IL-2 was obtained by FLP recombinase-mediated site-specific recombination. The amounts of GM-CSF and IL-2 in the culture supernatants at different time points were measured by enzyme-linked immunosorbent assay after human bone marrow mesenchymal stem cells were infected by Ad5 GM-CSF-IL-2 and irradiated with y-ray to lose proliferative activity. RESULTS AND CONCLUSION： The sequences of GM-CSF and IL-2 cDNAs were identical with those provided by GenBank NM_000758 （435 bp） and GenBank NM_000586 （462 bp） by sequencing, respectively. AdMaxTM was an efficient and quick packaging system of adenoviral vectors. The GM-CSF and IL-2 linked by IRES in the adenovirus can efficiently be expressed in the human bone marrow mesenchymal stem cetts at a high level for 7 days, suggesting a potential application to the tumor immunotherapy taking human bone marrow mesenchymal stem cells as a carrier expressing the activating factors for dendritic cells and T cells.