重组杆状病毒转导恒河猴骨髓间充质干细胞

Transduction of rhesus bone marrow mesenchymal stem cells by recombinant baculovirus

摘要:

【目的】研究重组杆状病毒(Bac-CMV-EGFP)能否能有效转导恒河猴骨髓间充质干细胞(rhesus Bone marrow-derived Mesenchymal Stem Cells, rBMSCs), 及杆状病毒转导后对细胞活力, 增殖及分化能力的影响。【方法】体外原代培养rBMSCs, 不同剂量的杆状病毒转导3代以后的细胞, 并用流式细胞仪分别检测其转导效率。在较高的杆状病毒转导效率下, 检测rBMSCs细胞活力, 增殖及分化 能力, 并与正常对照组细胞进行比较。【结果】杆状病毒在感染指数(Multiplicity Of Infection, MOI)为300v.g/cell, 孵育温度为25度, 孵育时间为4h的转导条件下, 对rBMSCs转导效率可达80％左右。进一步检测后发现, 高效转导杆状病毒后的rBMSCs的细胞活力, 增殖及分化能力与未转导病毒细胞组无明显变化。【结论】重组杆状病毒可安全有效地基因修饰rBMSCs, 且不影响其生物特性, 为 今后的体内基因治疗灵长类动物模型试验奠定了基础。

Abstract:

[Objective] To investigate whether the recombinant baculovirus (Bac-CMV-EGFP) can effectively transduce into rhesus Bone-marrow derived Mesenchymal Stem Cells (rBMSCs) in vitro, and whether there are some efficiency to the rBMSCs of viability, proliferational and differentiational capacity after recombinant baculovirus transducing. [Methods] The rBMSCs were cultured in vitro. After passaged more than three times, the rBMSCs were transduced with various dose of baculovirus (Multiplicity Of Infection, MOI, the MOI is 50, 100, 200, 300, and 500 vector genome (vg)/cell, re-spectively). We used flow cytometry to detect different transductive efficiency of various dose of baculovirus to rBMSCs. Under a suitable dose of baculovirus (300v.g/cell), we studied cell viability, proliferation and differentiation capacity, and compared results with the control. [Results] Baculovirus could be transduced into rBMSCs in vitro. The transductive ef-ficiency reached about 80% when the MOI was 300v.g/cell, at 25℃, and incubated for 4 h. Furthermore, under a higher transductive efficiency of baculovirus, there were no obvious influence to the rBMSCs of viability, proliferation and dif-ferentiation capacity compared with that of the control. [Conclusion] The baculovirus can be safely and effectively transduced into rBMSCs in vitro, without any negative efficiency to cell viability, proliferation and differentiation capacity.